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Structure (v.19, #8)

In This Issue (pp. v-vi).
In This Issue (pp. v-vi).
In This Issue (pp. v-vi).
In This Issue (pp. v-vi).

Mechanistic Insight from Chaos: How RecA Mediates DNA Strand Exchange by Claire Wyman (pp. 1031-1032).
Cleverly designed single-molecule FRET experiments reported in this issue of Structure by Ragunathan et al. coax RecA to reveal some of its secrets. Observing individual events identifies intermediate steps and provides clues for how to drive strand exchange forward.

Mechanistic Insight from Chaos: How RecA Mediates DNA Strand Exchange by Claire Wyman (pp. 1031-1032).
Cleverly designed single-molecule FRET experiments reported in this issue of Structure by Ragunathan et al. coax RecA to reveal some of its secrets. Observing individual events identifies intermediate steps and provides clues for how to drive strand exchange forward.

Mechanistic Insight from Chaos: How RecA Mediates DNA Strand Exchange by Claire Wyman (pp. 1031-1032).
Cleverly designed single-molecule FRET experiments reported in this issue of Structure by Ragunathan et al. coax RecA to reveal some of its secrets. Observing individual events identifies intermediate steps and provides clues for how to drive strand exchange forward.

Mechanistic Insight from Chaos: How RecA Mediates DNA Strand Exchange by Claire Wyman (pp. 1031-1032).
Cleverly designed single-molecule FRET experiments reported in this issue of Structure by Ragunathan et al. coax RecA to reveal some of its secrets. Observing individual events identifies intermediate steps and provides clues for how to drive strand exchange forward.

Nipped in the Bud: How the AMSH MIT Domain Helps Deubiquitinate Lysosome-Bound Cargo by James H. Hurley (pp. 1033-1035).
Recruitment of the K63-linkage specific deubiquitinating enzyme AMSH is an important step in ESCRT-dependent membrane protein sorting. In this issue of Structure, Solomons et al. now reveal an extraordinarily high affinity complex between the “MIM4” region of one ESCRT-III subunit, CHMP3, and the MIT domain of AMSH.

Nipped in the Bud: How the AMSH MIT Domain Helps Deubiquitinate Lysosome-Bound Cargo by James H. Hurley (pp. 1033-1035).
Recruitment of the K63-linkage specific deubiquitinating enzyme AMSH is an important step in ESCRT-dependent membrane protein sorting. In this issue of Structure, Solomons et al. now reveal an extraordinarily high affinity complex between the “MIM4” region of one ESCRT-III subunit, CHMP3, and the MIT domain of AMSH.

Nipped in the Bud: How the AMSH MIT Domain Helps Deubiquitinate Lysosome-Bound Cargo by James H. Hurley (pp. 1033-1035).
Recruitment of the K63-linkage specific deubiquitinating enzyme AMSH is an important step in ESCRT-dependent membrane protein sorting. In this issue of Structure, Solomons et al. now reveal an extraordinarily high affinity complex between the “MIM4” region of one ESCRT-III subunit, CHMP3, and the MIT domain of AMSH.

Nipped in the Bud: How the AMSH MIT Domain Helps Deubiquitinate Lysosome-Bound Cargo by James H. Hurley (pp. 1033-1035).
Recruitment of the K63-linkage specific deubiquitinating enzyme AMSH is an important step in ESCRT-dependent membrane protein sorting. In this issue of Structure, Solomons et al. now reveal an extraordinarily high affinity complex between the “MIM4” region of one ESCRT-III subunit, CHMP3, and the MIT domain of AMSH.

Bacterial, Fungal, and Algal Lectins: Combatants in Tug of War against HIV by Ten Feizi; Yan Liu; Angelina S. Palma (pp. 1035-1037).
High-resolution X-ray crystallography and NMR studies by Koharudin and Gronenborn in this issue provide new information on the mode of N-glycan recognition by a cyanobacterial agglutinin, with anti-HIV activity pointing to the pentamannosyl core as a novel target for therapeutic intervention.

Bacterial, Fungal, and Algal Lectins: Combatants in Tug of War against HIV by Ten Feizi; Yan Liu; Angelina S. Palma (pp. 1035-1037).
High-resolution X-ray crystallography and NMR studies by Koharudin and Gronenborn in this issue provide new information on the mode of N-glycan recognition by a cyanobacterial agglutinin, with anti-HIV activity pointing to the pentamannosyl core as a novel target for therapeutic intervention.

Bacterial, Fungal, and Algal Lectins: Combatants in Tug of War against HIV by Ten Feizi; Yan Liu; Angelina S. Palma (pp. 1035-1037).
High-resolution X-ray crystallography and NMR studies by Koharudin and Gronenborn in this issue provide new information on the mode of N-glycan recognition by a cyanobacterial agglutinin, with anti-HIV activity pointing to the pentamannosyl core as a novel target for therapeutic intervention.

Bacterial, Fungal, and Algal Lectins: Combatants in Tug of War against HIV by Ten Feizi; Yan Liu; Angelina S. Palma (pp. 1035-1037).
High-resolution X-ray crystallography and NMR studies by Koharudin and Gronenborn in this issue provide new information on the mode of N-glycan recognition by a cyanobacterial agglutinin, with anti-HIV activity pointing to the pentamannosyl core as a novel target for therapeutic intervention.

Insights into [FeFe]-Hydrogenase Structure, Mechanism, and Maturation by David W. Mulder; Eric M. Shepard; Jonathan E. Meuser; Neelambari Joshi; Paul W. King; Matthew C. Posewitz; Joan B. Broderick; John W. Peters (pp. 1038-1052).
Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.

Insights into [FeFe]-Hydrogenase Structure, Mechanism, and Maturation by David W. Mulder; Eric M. Shepard; Jonathan E. Meuser; Neelambari Joshi; Paul W. King; Matthew C. Posewitz; Joan B. Broderick; John W. Peters (pp. 1038-1052).
Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.

Insights into [FeFe]-Hydrogenase Structure, Mechanism, and Maturation by David W. Mulder; Eric M. Shepard; Jonathan E. Meuser; Neelambari Joshi; Paul W. King; Matthew C. Posewitz; Joan B. Broderick; John W. Peters (pp. 1038-1052).
Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.

Insights into [FeFe]-Hydrogenase Structure, Mechanism, and Maturation by David W. Mulder; Eric M. Shepard; Jonathan E. Meuser; Neelambari Joshi; Paul W. King; Matthew C. Posewitz; Joan B. Broderick; John W. Peters (pp. 1038-1052).
Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.

Preparation of Distinct Ubiquitin Chain Reagents of High Purity and Yield by Ken C. Dong; Elizabeth Helgason; Christine Yu; Lilian Phu; David P. Arnott; Ivan Bosanac; Deanne M. Compaan; Oscar W. Huang; Anna V. Fedorova; Donald S. Kirkpatrick; Sarah G. Hymowitz; Erin C. Dueber (pp. 1053-1063).
The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.Display Omitted► Methods for preparing multimilligram quantities of discrete ubiquitin chains ► Purifications and enzyme-driven chain syntheses use readily accessible protocols ► The high-quality ubiquitin chains can be used for biophysical/structural studies ► Methodology enables the incorporation of site-specific mutations and/or labels

Preparation of Distinct Ubiquitin Chain Reagents of High Purity and Yield by Ken C. Dong; Elizabeth Helgason; Christine Yu; Lilian Phu; David P. Arnott; Ivan Bosanac; Deanne M. Compaan; Oscar W. Huang; Anna V. Fedorova; Donald S. Kirkpatrick; Sarah G. Hymowitz; Erin C. Dueber (pp. 1053-1063).
The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.Display Omitted► Methods for preparing multimilligram quantities of discrete ubiquitin chains ► Purifications and enzyme-driven chain syntheses use readily accessible protocols ► The high-quality ubiquitin chains can be used for biophysical/structural studies ► Methodology enables the incorporation of site-specific mutations and/or labels

Preparation of Distinct Ubiquitin Chain Reagents of High Purity and Yield by Ken C. Dong; Elizabeth Helgason; Christine Yu; Lilian Phu; David P. Arnott; Ivan Bosanac; Deanne M. Compaan; Oscar W. Huang; Anna V. Fedorova; Donald S. Kirkpatrick; Sarah G. Hymowitz; Erin C. Dueber (pp. 1053-1063).
The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.Display Omitted► Methods for preparing multimilligram quantities of discrete ubiquitin chains ► Purifications and enzyme-driven chain syntheses use readily accessible protocols ► The high-quality ubiquitin chains can be used for biophysical/structural studies ► Methodology enables the incorporation of site-specific mutations and/or labels

Preparation of Distinct Ubiquitin Chain Reagents of High Purity and Yield by Ken C. Dong; Elizabeth Helgason; Christine Yu; Lilian Phu; David P. Arnott; Ivan Bosanac; Deanne M. Compaan; Oscar W. Huang; Anna V. Fedorova; Donald S. Kirkpatrick; Sarah G. Hymowitz; Erin C. Dueber (pp. 1053-1063).
The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.Display Omitted► Methods for preparing multimilligram quantities of discrete ubiquitin chains ► Purifications and enzyme-driven chain syntheses use readily accessible protocols ► The high-quality ubiquitin chains can be used for biophysical/structural studies ► Methodology enables the incorporation of site-specific mutations and/or labels

Real-Time Observation of Strand Exchange Reaction with High Spatiotemporal Resolution by Kaushik Ragunathan; Chirlmin Joo; Taekjip Ha (pp. 1064-1073).
RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.Display Omitted► Assay separates initial docking from subsequent propagation of RecA strand exchange ► Propagation occurs in approximately 3 bp increments ► Propagation occurs at a surprisingly high but physiologically relevant speed ► Discovered the formation of a dynamic complex between RecA and the displaced ssDNA

Real-Time Observation of Strand Exchange Reaction with High Spatiotemporal Resolution by Kaushik Ragunathan; Chirlmin Joo; Taekjip Ha (pp. 1064-1073).
RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.Display Omitted► Assay separates initial docking from subsequent propagation of RecA strand exchange ► Propagation occurs in approximately 3 bp increments ► Propagation occurs at a surprisingly high but physiologically relevant speed ► Discovered the formation of a dynamic complex between RecA and the displaced ssDNA

Real-Time Observation of Strand Exchange Reaction with High Spatiotemporal Resolution by Kaushik Ragunathan; Chirlmin Joo; Taekjip Ha (pp. 1064-1073).
RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.Display Omitted► Assay separates initial docking from subsequent propagation of RecA strand exchange ► Propagation occurs in approximately 3 bp increments ► Propagation occurs at a surprisingly high but physiologically relevant speed ► Discovered the formation of a dynamic complex between RecA and the displaced ssDNA

Real-Time Observation of Strand Exchange Reaction with High Spatiotemporal Resolution by Kaushik Ragunathan; Chirlmin Joo; Taekjip Ha (pp. 1064-1073).
RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.Display Omitted► Assay separates initial docking from subsequent propagation of RecA strand exchange ► Propagation occurs in approximately 3 bp increments ► Propagation occurs at a surprisingly high but physiologically relevant speed ► Discovered the formation of a dynamic complex between RecA and the displaced ssDNA

Crystal Structure of Full-Length Apaf-1: How the Death Signal Is Relayed in the Mitochondrial Pathway of Apoptosis by Thomas Frank Reubold; Sabine Wohlgemuth; Susanne Eschenburg (pp. 1074-1083).
The apoptotic protease-activating factor 1 (Apaf-1) relays the death signal in the mitochondrial pathway of apoptosis. Apaf-1 oligomerizes on binding of mitochondrially released cytochrome c into the heptameric apoptosome complex to ignite the downstream cascade of caspases. Here, we present the 3.0 Å crystal structure of full-length murine Apaf-1 in the absence of cytochrome c. The structure shows how the mammalian death switch is kept in its “off” position. By comparing the off state with a recent cryo-electron microscopy derived model of Apaf-1 in its apoptosomal conformation, we depict the molecular events that transform Apaf-1 from autoinhibited monomer to a building block of the caspase-activating apoptosome. Moreover, we have solved the crystal structure of the R265S mutant of full-length murine Apaf-1 in the absence of cytochrome c to 3.55 Å resolution and we show that proper function of Apaf-1 relies on R265 in the vicinity of the bound nucleotide.► Full-length structure of Apaf-1 shows the location of the regulatory domain ► Depiction of the molecular events that lead to apoptosome formation ► Identification of a residue essential for Apaf-1 function ► Explanation of how cytochrome c binding releases autoinhibition of Apaf-1

Crystal Structure of Full-Length Apaf-1: How the Death Signal Is Relayed in the Mitochondrial Pathway of Apoptosis by Thomas Frank Reubold; Sabine Wohlgemuth; Susanne Eschenburg (pp. 1074-1083).
The apoptotic protease-activating factor 1 (Apaf-1) relays the death signal in the mitochondrial pathway of apoptosis. Apaf-1 oligomerizes on binding of mitochondrially released cytochrome c into the heptameric apoptosome complex to ignite the downstream cascade of caspases. Here, we present the 3.0 Å crystal structure of full-length murine Apaf-1 in the absence of cytochrome c. The structure shows how the mammalian death switch is kept in its “off” position. By comparing the off state with a recent cryo-electron microscopy derived model of Apaf-1 in its apoptosomal conformation, we depict the molecular events that transform Apaf-1 from autoinhibited monomer to a building block of the caspase-activating apoptosome. Moreover, we have solved the crystal structure of the R265S mutant of full-length murine Apaf-1 in the absence of cytochrome c to 3.55 Å resolution and we show that proper function of Apaf-1 relies on R265 in the vicinity of the bound nucleotide.► Full-length structure of Apaf-1 shows the location of the regulatory domain ► Depiction of the molecular events that lead to apoptosome formation ► Identification of a residue essential for Apaf-1 function ► Explanation of how cytochrome c binding releases autoinhibition of Apaf-1

Crystal Structure of Full-Length Apaf-1: How the Death Signal Is Relayed in the Mitochondrial Pathway of Apoptosis by Thomas Frank Reubold; Sabine Wohlgemuth; Susanne Eschenburg (pp. 1074-1083).
The apoptotic protease-activating factor 1 (Apaf-1) relays the death signal in the mitochondrial pathway of apoptosis. Apaf-1 oligomerizes on binding of mitochondrially released cytochrome c into the heptameric apoptosome complex to ignite the downstream cascade of caspases. Here, we present the 3.0 Å crystal structure of full-length murine Apaf-1 in the absence of cytochrome c. The structure shows how the mammalian death switch is kept in its “off” position. By comparing the off state with a recent cryo-electron microscopy derived model of Apaf-1 in its apoptosomal conformation, we depict the molecular events that transform Apaf-1 from autoinhibited monomer to a building block of the caspase-activating apoptosome. Moreover, we have solved the crystal structure of the R265S mutant of full-length murine Apaf-1 in the absence of cytochrome c to 3.55 Å resolution and we show that proper function of Apaf-1 relies on R265 in the vicinity of the bound nucleotide.► Full-length structure of Apaf-1 shows the location of the regulatory domain ► Depiction of the molecular events that lead to apoptosome formation ► Identification of a residue essential for Apaf-1 function ► Explanation of how cytochrome c binding releases autoinhibition of Apaf-1

Crystal Structure of Full-Length Apaf-1: How the Death Signal Is Relayed in the Mitochondrial Pathway of Apoptosis by Thomas Frank Reubold; Sabine Wohlgemuth; Susanne Eschenburg (pp. 1074-1083).
The apoptotic protease-activating factor 1 (Apaf-1) relays the death signal in the mitochondrial pathway of apoptosis. Apaf-1 oligomerizes on binding of mitochondrially released cytochrome c into the heptameric apoptosome complex to ignite the downstream cascade of caspases. Here, we present the 3.0 Å crystal structure of full-length murine Apaf-1 in the absence of cytochrome c. The structure shows how the mammalian death switch is kept in its “off” position. By comparing the off state with a recent cryo-electron microscopy derived model of Apaf-1 in its apoptosomal conformation, we depict the molecular events that transform Apaf-1 from autoinhibited monomer to a building block of the caspase-activating apoptosome. Moreover, we have solved the crystal structure of the R265S mutant of full-length murine Apaf-1 in the absence of cytochrome c to 3.55 Å resolution and we show that proper function of Apaf-1 relies on R265 in the vicinity of the bound nucleotide.► Full-length structure of Apaf-1 shows the location of the regulatory domain ► Depiction of the molecular events that lead to apoptosome formation ► Identification of a residue essential for Apaf-1 function ► Explanation of how cytochrome c binding releases autoinhibition of Apaf-1

The Holo-Apoptosome: Activation of Procaspase-9 and Interactions with Caspase-3 by Shujun Yuan; Xinchao Yu; John M. Asara; John E. Heuser; Steven J. Ludtke; Christopher W. Akey (pp. 1084-1096).
Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.► A mechanism of proximity-induced association is proposed for procaspase-9 activation ► Coassembly of a CARD-CARD disk creates an asymmetric proteolysis machine ► Procaspase-3 and -9 share overlapping binding sites on the human apoptosome ► Caspase-3 may displace pc-9 from the apoptosome to create an executioner complex

The Holo-Apoptosome: Activation of Procaspase-9 and Interactions with Caspase-3 by Shujun Yuan; Xinchao Yu; John M. Asara; John E. Heuser; Steven J. Ludtke; Christopher W. Akey (pp. 1084-1096).
Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.► A mechanism of proximity-induced association is proposed for procaspase-9 activation ► Coassembly of a CARD-CARD disk creates an asymmetric proteolysis machine ► Procaspase-3 and -9 share overlapping binding sites on the human apoptosome ► Caspase-3 may displace pc-9 from the apoptosome to create an executioner complex

The Holo-Apoptosome: Activation of Procaspase-9 and Interactions with Caspase-3 by Shujun Yuan; Xinchao Yu; John M. Asara; John E. Heuser; Steven J. Ludtke; Christopher W. Akey (pp. 1084-1096).
Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.► A mechanism of proximity-induced association is proposed for procaspase-9 activation ► Coassembly of a CARD-CARD disk creates an asymmetric proteolysis machine ► Procaspase-3 and -9 share overlapping binding sites on the human apoptosome ► Caspase-3 may displace pc-9 from the apoptosome to create an executioner complex

The Holo-Apoptosome: Activation of Procaspase-9 and Interactions with Caspase-3 by Shujun Yuan; Xinchao Yu; John M. Asara; John E. Heuser; Steven J. Ludtke; Christopher W. Akey (pp. 1084-1096).
Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.► A mechanism of proximity-induced association is proposed for procaspase-9 activation ► Coassembly of a CARD-CARD disk creates an asymmetric proteolysis machine ► Procaspase-3 and -9 share overlapping binding sites on the human apoptosome ► Caspase-3 may displace pc-9 from the apoptosome to create an executioner complex

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies by Matthew C. Franklin; Elizabeth C. Navarro; Yujie Wang; Sheetal Patel; Pinki Singh; Yi Zhang; Kris Persaud; Amtul Bari; Heather Griffith; Leyi Shen; Paul Balderes; Paul Kussie (pp. 1097-1107).
The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.► 1121B and 6.64 both block VEGF binding to KDR, but do so in completely different ways ► CDR H2 in 1121B sterically clashes with a loop in bound VEGF-C (and probably VEGF-A ► 6.64 blocks VEGF binding to AP-tagged KDR, but not to the Fc-tagged equivalent ► The 1121B and VEGF contact surfaces on KDR domain 3 abut, but do not overlap

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies by Matthew C. Franklin; Elizabeth C. Navarro; Yujie Wang; Sheetal Patel; Pinki Singh; Yi Zhang; Kris Persaud; Amtul Bari; Heather Griffith; Leyi Shen; Paul Balderes; Paul Kussie (pp. 1097-1107).
The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.► 1121B and 6.64 both block VEGF binding to KDR, but do so in completely different ways ► CDR H2 in 1121B sterically clashes with a loop in bound VEGF-C (and probably VEGF-A ► 6.64 blocks VEGF binding to AP-tagged KDR, but not to the Fc-tagged equivalent ► The 1121B and VEGF contact surfaces on KDR domain 3 abut, but do not overlap

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies by Matthew C. Franklin; Elizabeth C. Navarro; Yujie Wang; Sheetal Patel; Pinki Singh; Yi Zhang; Kris Persaud; Amtul Bari; Heather Griffith; Leyi Shen; Paul Balderes; Paul Kussie (pp. 1097-1107).
The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.► 1121B and 6.64 both block VEGF binding to KDR, but do so in completely different ways ► CDR H2 in 1121B sterically clashes with a loop in bound VEGF-C (and probably VEGF-A ► 6.64 blocks VEGF binding to AP-tagged KDR, but not to the Fc-tagged equivalent ► The 1121B and VEGF contact surfaces on KDR domain 3 abut, but do not overlap

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies by Matthew C. Franklin; Elizabeth C. Navarro; Yujie Wang; Sheetal Patel; Pinki Singh; Yi Zhang; Kris Persaud; Amtul Bari; Heather Griffith; Leyi Shen; Paul Balderes; Paul Kussie (pp. 1097-1107).
The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.► 1121B and 6.64 both block VEGF binding to KDR, but do so in completely different ways ► CDR H2 in 1121B sterically clashes with a loop in bound VEGF-C (and probably VEGF-A ► 6.64 blocks VEGF binding to AP-tagged KDR, but not to the Fc-tagged equivalent ► The 1121B and VEGF contact surfaces on KDR domain 3 abut, but do not overlap

Status of GPCR Modeling and Docking as Reflected by Community-wide GPCR Dock 2010 Assessment by Irina Kufareva; Manuel Rueda; Vsevolod Katritch; Raymond C. Stevens; Ruben Abagyan (pp. 1108-1126).
The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G protein-coupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast, CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.Display Omitted► The GPCR Dock 2010 assessment featured three targets of varying modeling difficulty ► Thirty-five groups submitted 275 GPCR complex models prior to release of X-ray coordinates ► Best predictions capture GPCR-ligand interaction details at atomic resolution level ► Reliable homology modeling requires 35%–40% sequence identity between target and template

Status of GPCR Modeling and Docking as Reflected by Community-wide GPCR Dock 2010 Assessment by Irina Kufareva; Manuel Rueda; Vsevolod Katritch; Raymond C. Stevens; Ruben Abagyan (pp. 1108-1126).
The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G protein-coupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast, CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.Display Omitted► The GPCR Dock 2010 assessment featured three targets of varying modeling difficulty ► Thirty-five groups submitted 275 GPCR complex models prior to release of X-ray coordinates ► Best predictions capture GPCR-ligand interaction details at atomic resolution level ► Reliable homology modeling requires 35%–40% sequence identity between target and template

Status of GPCR Modeling and Docking as Reflected by Community-wide GPCR Dock 2010 Assessment by Irina Kufareva; Manuel Rueda; Vsevolod Katritch; Raymond C. Stevens; Ruben Abagyan (pp. 1108-1126).
The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G protein-coupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast, CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.Display Omitted► The GPCR Dock 2010 assessment featured three targets of varying modeling difficulty ► Thirty-five groups submitted 275 GPCR complex models prior to release of X-ray coordinates ► Best predictions capture GPCR-ligand interaction details at atomic resolution level ► Reliable homology modeling requires 35%–40% sequence identity between target and template

Status of GPCR Modeling and Docking as Reflected by Community-wide GPCR Dock 2010 Assessment by Irina Kufareva; Manuel Rueda; Vsevolod Katritch; Raymond C. Stevens; Ruben Abagyan (pp. 1108-1126).
The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G protein-coupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast, CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.Display Omitted► The GPCR Dock 2010 assessment featured three targets of varying modeling difficulty ► Thirty-five groups submitted 275 GPCR complex models prior to release of X-ray coordinates ► Best predictions capture GPCR-ligand interaction details at atomic resolution level ► Reliable homology modeling requires 35%–40% sequence identity between target and template

Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α by John E. Burke; Oscar Vadas; Alex Berndt; Tara Finegan; Olga Perisic; Roger L. Williams (pp. 1127-1137).
Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.► Both nSH2 and cSH2 of p85α inhibit p110δ, and pY peptides or mutations activate ► DXMS mapped dynamic changes in free p110δ and p110δ/p85 with and without pY peptides ► pY peptide increases p110δ/p85 affinity for lipids; interactions were mapped by DXMS ► cSH2 point mutation in p85α activates p110δ and p110β, but not p110α

Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α by John E. Burke; Oscar Vadas; Alex Berndt; Tara Finegan; Olga Perisic; Roger L. Williams (pp. 1127-1137).
Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.► Both nSH2 and cSH2 of p85α inhibit p110δ, and pY peptides or mutations activate ► DXMS mapped dynamic changes in free p110δ and p110δ/p85 with and without pY peptides ► pY peptide increases p110δ/p85 affinity for lipids; interactions were mapped by DXMS ► cSH2 point mutation in p85α activates p110δ and p110β, but not p110α

Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α by John E. Burke; Oscar Vadas; Alex Berndt; Tara Finegan; Olga Perisic; Roger L. Williams (pp. 1127-1137).
Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.► Both nSH2 and cSH2 of p85α inhibit p110δ, and pY peptides or mutations activate ► DXMS mapped dynamic changes in free p110δ and p110δ/p85 with and without pY peptides ► pY peptide increases p110δ/p85 affinity for lipids; interactions were mapped by DXMS ► cSH2 point mutation in p85α activates p110δ and p110β, but not p110α

Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α by John E. Burke; Oscar Vadas; Alex Berndt; Tara Finegan; Olga Perisic; Roger L. Williams (pp. 1127-1137).
Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.► Both nSH2 and cSH2 of p85α inhibit p110δ, and pY peptides or mutations activate ► DXMS mapped dynamic changes in free p110δ and p110δ/p85 with and without pY peptides ► pY peptide increases p110δ/p85 affinity for lipids; interactions were mapped by DXMS ► cSH2 point mutation in p85α activates p110δ and p110β, but not p110α

Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data by Xu Wang; Caroline Watson; Joshua S. Sharp; Tracy M. Handel; James H. Prestegard (pp. 1138-1148).
CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo.Display Omitted► Wild-type CCL5 oligomerizes to even order structures in a pH-dependent manner ► A tetramer structure was determined by integrating NMR, SAXS, and MS data ► Higher-order oligomers are formed by dimers ordered in a staggered line ► The model exposes GAG- and CCR5-binding residues, for simultaneous interaction

Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data by Xu Wang; Caroline Watson; Joshua S. Sharp; Tracy M. Handel; James H. Prestegard (pp. 1138-1148).
CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo.Display Omitted► Wild-type CCL5 oligomerizes to even order structures in a pH-dependent manner ► A tetramer structure was determined by integrating NMR, SAXS, and MS data ► Higher-order oligomers are formed by dimers ordered in a staggered line ► The model exposes GAG- and CCR5-binding residues, for simultaneous interaction

Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data by Xu Wang; Caroline Watson; Joshua S. Sharp; Tracy M. Handel; James H. Prestegard (pp. 1138-1148).
CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo.Display Omitted► Wild-type CCL5 oligomerizes to even order structures in a pH-dependent manner ► A tetramer structure was determined by integrating NMR, SAXS, and MS data ► Higher-order oligomers are formed by dimers ordered in a staggered line ► The model exposes GAG- and CCR5-binding residues, for simultaneous interaction

Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data by Xu Wang; Caroline Watson; Joshua S. Sharp; Tracy M. Handel; James H. Prestegard (pp. 1138-1148).
CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo.Display Omitted► Wild-type CCL5 oligomerizes to even order structures in a pH-dependent manner ► A tetramer structure was determined by integrating NMR, SAXS, and MS data ► Higher-order oligomers are formed by dimers ordered in a staggered line ► The model exposes GAG- and CCR5-binding residues, for simultaneous interaction

Structural Basis for ESCRT-III CHMP3 Recruitment of AMSH by Julianna Solomons; Charles Sabin; Emilie Poudevigne; Yoshiko Usami; David Lutje Hulsik; Pauline Macheboeuf; Bettina Hartlieb; Heinrich Göttlinger; Winfried Weissenhorn (pp. 1149-1159).
Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.Display Omitted► The N-terminal protease resistant core of AMSH contains an unusual MIT domain, with the MIT helix 3 homologue extending to 90 Å ► The last C-terminal 20 amino acids of CHMP3 are unstructured in solution and adopt a helical conformation in complex with the AMSH MIT domain ► CHMP3 binds with nanomolar affinity to AMSH ► The N-terminal region of AMSH preceding the MIT domain reveals conformational flexibility that might be important for AMSH regulation during HIV-1 budding

Structural Basis for ESCRT-III CHMP3 Recruitment of AMSH by Julianna Solomons; Charles Sabin; Emilie Poudevigne; Yoshiko Usami; David Lutje Hulsik; Pauline Macheboeuf; Bettina Hartlieb; Heinrich Göttlinger; Winfried Weissenhorn (pp. 1149-1159).
Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.Display Omitted► The N-terminal protease resistant core of AMSH contains an unusual MIT domain, with the MIT helix 3 homologue extending to 90 Å ► The last C-terminal 20 amino acids of CHMP3 are unstructured in solution and adopt a helical conformation in complex with the AMSH MIT domain ► CHMP3 binds with nanomolar affinity to AMSH ► The N-terminal region of AMSH preceding the MIT domain reveals conformational flexibility that might be important for AMSH regulation during HIV-1 budding

Structural Basis for ESCRT-III CHMP3 Recruitment of AMSH by Julianna Solomons; Charles Sabin; Emilie Poudevigne; Yoshiko Usami; David Lutje Hulsik; Pauline Macheboeuf; Bettina Hartlieb; Heinrich Göttlinger; Winfried Weissenhorn (pp. 1149-1159).
Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.Display Omitted► The N-terminal protease resistant core of AMSH contains an unusual MIT domain, with the MIT helix 3 homologue extending to 90 Å ► The last C-terminal 20 amino acids of CHMP3 are unstructured in solution and adopt a helical conformation in complex with the AMSH MIT domain ► CHMP3 binds with nanomolar affinity to AMSH ► The N-terminal region of AMSH preceding the MIT domain reveals conformational flexibility that might be important for AMSH regulation during HIV-1 budding

Structural Basis for ESCRT-III CHMP3 Recruitment of AMSH by Julianna Solomons; Charles Sabin; Emilie Poudevigne; Yoshiko Usami; David Lutje Hulsik; Pauline Macheboeuf; Bettina Hartlieb; Heinrich Göttlinger; Winfried Weissenhorn (pp. 1149-1159).
Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.Display Omitted► The N-terminal protease resistant core of AMSH contains an unusual MIT domain, with the MIT helix 3 homologue extending to 90 Å ► The last C-terminal 20 amino acids of CHMP3 are unstructured in solution and adopt a helical conformation in complex with the AMSH MIT domain ► CHMP3 binds with nanomolar affinity to AMSH ► The N-terminal region of AMSH preceding the MIT domain reveals conformational flexibility that might be important for AMSH regulation during HIV-1 budding

Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease by Masayoshi Sakakura; Arina Hadziselimovic; Zhen Wang; Kevin L. Schey; Charles R. Sanders (pp. 1160-1169).
Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state, transmembrane helices 2–4 (TM2–4) form a molten globular bundle, while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2–4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein-folding quality control, which leads to loss of function and toxic accumulation of aggregates that result in CMTD.Display Omitted► The Trembler-J (Leu16Pro) mutant form of peripheral myelin protein 22 was characterized ► The TrJ mutation profoundly alters the structure/dynamics of TM segment 1 ► Defects in TM1 lead to destabilization of PMP22 and dissociation of TM1 from TM2–4 ► The above defects lead to misfolding of PMP22 and Charcot-Marie-Tooth disease

Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease by Masayoshi Sakakura; Arina Hadziselimovic; Zhen Wang; Kevin L. Schey; Charles R. Sanders (pp. 1160-1169).
Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state, transmembrane helices 2–4 (TM2–4) form a molten globular bundle, while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2–4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein-folding quality control, which leads to loss of function and toxic accumulation of aggregates that result in CMTD.Display Omitted► The Trembler-J (Leu16Pro) mutant form of peripheral myelin protein 22 was characterized ► The TrJ mutation profoundly alters the structure/dynamics of TM segment 1 ► Defects in TM1 lead to destabilization of PMP22 and dissociation of TM1 from TM2–4 ► The above defects lead to misfolding of PMP22 and Charcot-Marie-Tooth disease

Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease by Masayoshi Sakakura; Arina Hadziselimovic; Zhen Wang; Kevin L. Schey; Charles R. Sanders (pp. 1160-1169).
Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state, transmembrane helices 2–4 (TM2–4) form a molten globular bundle, while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2–4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein-folding quality control, which leads to loss of function and toxic accumulation of aggregates that result in CMTD.Display Omitted► The Trembler-J (Leu16Pro) mutant form of peripheral myelin protein 22 was characterized ► The TrJ mutation profoundly alters the structure/dynamics of TM segment 1 ► Defects in TM1 lead to destabilization of PMP22 and dissociation of TM1 from TM2–4 ► The above defects lead to misfolding of PMP22 and Charcot-Marie-Tooth disease

Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease by Masayoshi Sakakura; Arina Hadziselimovic; Zhen Wang; Kevin L. Schey; Charles R. Sanders (pp. 1160-1169).
Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state, transmembrane helices 2–4 (TM2–4) form a molten globular bundle, while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2–4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein-folding quality control, which leads to loss of function and toxic accumulation of aggregates that result in CMTD.Display Omitted► The Trembler-J (Leu16Pro) mutant form of peripheral myelin protein 22 was characterized ► The TrJ mutation profoundly alters the structure/dynamics of TM segment 1 ► Defects in TM1 lead to destabilization of PMP22 and dissociation of TM1 from TM2–4 ► The above defects lead to misfolding of PMP22 and Charcot-Marie-Tooth disease

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin by Leonardus M.I. Koharudin; Angela M. Gronenborn (pp. 1170-1181).
The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77–G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.► α3,α6-mannopentaose binds to OAA tightly and specifically at two binding sites ► High resolution structures of free and α3,α6-mannopentaose-bound OAA are presented ► The carbohydrate recognition of OAA is unique in comparison to other antiviral lectins ► Our results provide structural insights into the anti-HIV activity of OAA

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin by Leonardus M.I. Koharudin; Angela M. Gronenborn (pp. 1170-1181).
The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77–G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.► α3,α6-mannopentaose binds to OAA tightly and specifically at two binding sites ► High resolution structures of free and α3,α6-mannopentaose-bound OAA are presented ► The carbohydrate recognition of OAA is unique in comparison to other antiviral lectins ► Our results provide structural insights into the anti-HIV activity of OAA

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin by Leonardus M.I. Koharudin; Angela M. Gronenborn (pp. 1170-1181).
The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77–G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.► α3,α6-mannopentaose binds to OAA tightly and specifically at two binding sites ► High resolution structures of free and α3,α6-mannopentaose-bound OAA are presented ► The carbohydrate recognition of OAA is unique in comparison to other antiviral lectins ► Our results provide structural insights into the anti-HIV activity of OAA

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin by Leonardus M.I. Koharudin; Angela M. Gronenborn (pp. 1170-1181).
The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77–G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.► α3,α6-mannopentaose binds to OAA tightly and specifically at two binding sites ► High resolution structures of free and α3,α6-mannopentaose-bound OAA are presented ► The carbohydrate recognition of OAA is unique in comparison to other antiviral lectins ► Our results provide structural insights into the anti-HIV activity of OAA

Improving Protein Structure Prediction Using Multiple Sequence-Based Contact Predictions by Sitao Wu; Andras Szilagyi; Yang Zhang (pp. 1182-1191).
Although residue-residue contact maps dictate the topology of proteins, sequence-based ab initio contact predictions have been found little use in actual structure prediction due to the low accuracy. We developed a composite set of nine SVM-based contact predictors that are used in I-TASSER simulation in combination with sparse template contact restraints. When testing the strategy on 273 nonhomologous targets, remarkable improvements of I-TASSER models were observed for both easy and hard targets, with p value by Student's t test <0.00001 and 0.001, respectively. In several cases, template modeling score increases by >30%, which essentially converts “nonfoldable” targets into “foldable” ones. In CASP9, I-TASSER employed ab initio contact predictions, and generated models for 26 FM targets with a GDT-score 16% and 44% higher than the second and third best servers from other groups, respectively. These findings demonstrate a new avenue to improve the accuracy of protein structure prediction especially for free-modeling targets.Display Omitted► Ab initio contacts is demonstrated useful to protein structure prediction ► New method to combine protein structure prediction with contact predictions ► Remarkable improvement in protein structure prediction for easy and hard targets ► “Nonfoldable” targets can be converted to “foldable” ones

Improving Protein Structure Prediction Using Multiple Sequence-Based Contact Predictions by Sitao Wu; Andras Szilagyi; Yang Zhang (pp. 1182-1191).
Although residue-residue contact maps dictate the topology of proteins, sequence-based ab initio contact predictions have been found little use in actual structure prediction due to the low accuracy. We developed a composite set of nine SVM-based contact predictors that are used in I-TASSER simulation in combination with sparse template contact restraints. When testing the strategy on 273 nonhomologous targets, remarkable improvements of I-TASSER models were observed for both easy and hard targets, with p value by Student's t test <0.00001 and 0.001, respectively. In several cases, template modeling score increases by >30%, which essentially converts “nonfoldable” targets into “foldable” ones. In CASP9, I-TASSER employed ab initio contact predictions, and generated models for 26 FM targets with a GDT-score 16% and 44% higher than the second and third best servers from other groups, respectively. These findings demonstrate a new avenue to improve the accuracy of protein structure prediction especially for free-modeling targets.Display Omitted► Ab initio contacts is demonstrated useful to protein structure prediction ► New method to combine protein structure prediction with contact predictions ► Remarkable improvement in protein structure prediction for easy and hard targets ► “Nonfoldable” targets can be converted to “foldable” ones

Improving Protein Structure Prediction Using Multiple Sequence-Based Contact Predictions by Sitao Wu; Andras Szilagyi; Yang Zhang (pp. 1182-1191).
Although residue-residue contact maps dictate the topology of proteins, sequence-based ab initio contact predictions have been found little use in actual structure prediction due to the low accuracy. We developed a composite set of nine SVM-based contact predictors that are used in I-TASSER simulation in combination with sparse template contact restraints. When testing the strategy on 273 nonhomologous targets, remarkable improvements of I-TASSER models were observed for both easy and hard targets, with p value by Student's t test <0.00001 and 0.001, respectively. In several cases, template modeling score increases by >30%, which essentially converts “nonfoldable” targets into “foldable” ones. In CASP9, I-TASSER employed ab initio contact predictions, and generated models for 26 FM targets with a GDT-score 16% and 44% higher than the second and third best servers from other groups, respectively. These findings demonstrate a new avenue to improve the accuracy of protein structure prediction especially for free-modeling targets.Display Omitted► Ab initio contacts is demonstrated useful to protein structure prediction ► New method to combine protein structure prediction with contact predictions ► Remarkable improvement in protein structure prediction for easy and hard targets ► “Nonfoldable” targets can be converted to “foldable” ones

Improving Protein Structure Prediction Using Multiple Sequence-Based Contact Predictions by Sitao Wu; Andras Szilagyi; Yang Zhang (pp. 1182-1191).
Although residue-residue contact maps dictate the topology of proteins, sequence-based ab initio contact predictions have been found little use in actual structure prediction due to the low accuracy. We developed a composite set of nine SVM-based contact predictors that are used in I-TASSER simulation in combination with sparse template contact restraints. When testing the strategy on 273 nonhomologous targets, remarkable improvements of I-TASSER models were observed for both easy and hard targets, with p value by Student's t test <0.00001 and 0.001, respectively. In several cases, template modeling score increases by >30%, which essentially converts “nonfoldable” targets into “foldable” ones. In CASP9, I-TASSER employed ab initio contact predictions, and generated models for 26 FM targets with a GDT-score 16% and 44% higher than the second and third best servers from other groups, respectively. These findings demonstrate a new avenue to improve the accuracy of protein structure prediction especially for free-modeling targets.Display Omitted► Ab initio contacts is demonstrated useful to protein structure prediction ► New method to combine protein structure prediction with contact predictions ► Remarkable improvement in protein structure prediction for easy and hard targets ► “Nonfoldable” targets can be converted to “foldable” ones

Flexible Architecture of IP3R1 by Cryo-EM by Steven J. Ludtke; Thao P. Tran; Que T. Ngo; Vera Yu. Moiseenkova-Bell; Wah Chiu; Irina I. Serysheva (pp. 1192-1199).
Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.► 3D cryo-EM structure of IP3R in the closed state at subnanometer resolution ► Molecular architecture of ion-channel conduction pore ► Discovery of turret densities on the channel's lumenal surface ► Structural flexibility of the CY region in the absence of IP3 and Ca2+

Flexible Architecture of IP3R1 by Cryo-EM by Steven J. Ludtke; Thao P. Tran; Que T. Ngo; Vera Yu. Moiseenkova-Bell; Wah Chiu; Irina I. Serysheva (pp. 1192-1199).
Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.► 3D cryo-EM structure of IP3R in the closed state at subnanometer resolution ► Molecular architecture of ion-channel conduction pore ► Discovery of turret densities on the channel's lumenal surface ► Structural flexibility of the CY region in the absence of IP3 and Ca2+

Flexible Architecture of IP3R1 by Cryo-EM by Steven J. Ludtke; Thao P. Tran; Que T. Ngo; Vera Yu. Moiseenkova-Bell; Wah Chiu; Irina I. Serysheva (pp. 1192-1199).
Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.► 3D cryo-EM structure of IP3R in the closed state at subnanometer resolution ► Molecular architecture of ion-channel conduction pore ► Discovery of turret densities on the channel's lumenal surface ► Structural flexibility of the CY region in the absence of IP3 and Ca2+

Flexible Architecture of IP3R1 by Cryo-EM by Steven J. Ludtke; Thao P. Tran; Que T. Ngo; Vera Yu. Moiseenkova-Bell; Wah Chiu; Irina I. Serysheva (pp. 1192-1199).
Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.► 3D cryo-EM structure of IP3R in the closed state at subnanometer resolution ► Molecular architecture of ion-channel conduction pore ► Discovery of turret densities on the channel's lumenal surface ► Structural flexibility of the CY region in the absence of IP3 and Ca2+
Retraction Notice to: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer G12 by Christopher A. Johnston; Adam J. Kimple; Patrick M. Gigu re; David P. (pp. 1200-1200).
Retraction Notice to: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer G12 by Christopher A. Johnston; Adam J. Kimple; Patrick M. Gigu re; David P. (pp. 1200-1200).
Retraction Notice to: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer Gβ1γ2 by Christopher A. Johnston; Adam J. Kimple; Patrick M. Giguère; David P. Siderovski (pp. 1200-1200).

Retraction Notice to: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer G12 by Christopher A. Johnston; Adam J. Kimple; Patrick M. Gigu re; David P. (pp. 1200-1200).
(Structure 16, 10861094; July 9, 2008)In this paper, a cocrystal structure was described of the G-protein heterodimer G12 bound to a C-terminal peptide from the parathyroid hormone receptor-1 (PTH1R) at 3.0 resolution (PDB id 2QNS). A subsequent refinement was later deposited in the Protein Data Bank (PDB id 3KJ5). While this structure represents a new crystal form of the G12 heterodimer, because of the lack of clear and continuous electron density for the receptor peptide in the complex structure, the paper is being retracted. We apologize for any confusion this may have caused.
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