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Structure (v.18, #12)

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

FHA Domain pThr Binding Specificity: It's All about Me by Nicolas Coquelle; J.N. Mark Glover (pp. 1549-1550).
The FHA domain is a phospho-peptide binding module involved in a wide range of cellular pathways, with a striking specificity for phospho-threonine over phospho-serine binding partners. Biochemical, structural, and dynamic simulations analysis allowed Pennell and colleagues to unravel the molecular basis of FHA domain phospho-threonine specificity.

FHA Domain pThr Binding Specificity: It's All about Me by Nicolas Coquelle; J.N. Mark Glover (pp. 1549-1550).
The FHA domain is a phospho-peptide binding module involved in a wide range of cellular pathways, with a striking specificity for phospho-threonine over phospho-serine binding partners. Biochemical, structural, and dynamic simulations analysis allowed Pennell and colleagues to unravel the molecular basis of FHA domain phospho-threonine specificity.

Finding the Path in an RNA Folding Landscape by Sherry N. Boodram; Philip E. Johnson (pp. 1550-1551).
In this issue of Structure, combine molecular and computational biology approaches to provide structural details for intermediates in the folding pathway of the hepatitis delta virus ribozyme.

Finding the Path in an RNA Folding Landscape by Sherry N. Boodram; Philip E. Johnson (pp. 1550-1551).
In this issue of Structure, combine molecular and computational biology approaches to provide structural details for intermediates in the folding pathway of the hepatitis delta virus ribozyme.

Toward a Structural Understanding of Arf Family:Effector Specificity by Philippe Chavrier; Julie Ménétrey (pp. 1552-1558).
Arf family proteins are critical regulators of intracellular trafficking and actin cytoskeleton dynamics. To carry out their cellular functions, Arf family proteins interact with various effectors that differ in nature and structure. Understanding how these proteins interact with structurally different partners and are distinguished by specific effectors while being closely related requires a structural characterization and comparison of the various Arf family:effector complexes. Recent structural reports of Arf and Arl proteins in complex with different downstream effectors shed new light on general and specific structural recognition determinants characteristic of Arf family proteins.

Toward a Structural Understanding of Arf Family:Effector Specificity by Philippe Chavrier; Julie Ménétrey (pp. 1552-1558).
Arf family proteins are critical regulators of intracellular trafficking and actin cytoskeleton dynamics. To carry out their cellular functions, Arf family proteins interact with various effectors that differ in nature and structure. Understanding how these proteins interact with structurally different partners and are distinguished by specific effectors while being closely related requires a structural characterization and comparison of the various Arf family:effector complexes. Recent structural reports of Arf and Arl proteins in complex with different downstream effectors shed new light on general and specific structural recognition determinants characteristic of Arf family proteins.

A View into the Blind Spot: Solution NMR Provides New Insights into Signal Transduction Across the Lipid Bilayer by Matthew E. Call; James J. Chou (pp. 1559-1569).
One of the most fundamental problems in cell biology concerns how cells communicate with their surroundings through surface receptors. The last few decades have seen major advances in understanding the mechanisms of receptor-ligand recognition and the biochemical consequences of such encounters. This review describes the emergence of solution nuclear magnetic resonance (NMR) spectroscopy as a powerful tool for the structural characterization of membrane-associated protein domains involved in transmembrane signaling. We highlight particularly instructive examples from the fields of immunoreceptor biology, growth hormone signaling, and cell adhesion. These signaling complexes comprise multiple subunits each spanning the membrane with a single helical segment that links extracellular ligand-binding domains to the cell interior. The apparent simplicity of this domain organization belies the complexity involved in cooperative assembly of functional structures that translate information across the cellular boundary.

A View into the Blind Spot: Solution NMR Provides New Insights into Signal Transduction Across the Lipid Bilayer by Matthew E. Call; James J. Chou (pp. 1559-1569).
One of the most fundamental problems in cell biology concerns how cells communicate with their surroundings through surface receptors. The last few decades have seen major advances in understanding the mechanisms of receptor-ligand recognition and the biochemical consequences of such encounters. This review describes the emergence of solution nuclear magnetic resonance (NMR) spectroscopy as a powerful tool for the structural characterization of membrane-associated protein domains involved in transmembrane signaling. We highlight particularly instructive examples from the fields of immunoreceptor biology, growth hormone signaling, and cell adhesion. These signaling complexes comprise multiple subunits each spanning the membrane with a single helical segment that links extracellular ligand-binding domains to the cell interior. The apparent simplicity of this domain organization belies the complexity involved in cooperative assembly of functional structures that translate information across the cellular boundary.

The Third Conformation of p38α MAP Kinase Observed in Phosphorylated p38α and in Solution by Radha Akella; Xiaoshan Min; Qiong Wu; Kevin H. Gardner; Elizabeth J. Goldsmith (pp. 1571-1578).
MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T∗GY∗/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T∗GY∗) and ERK2 (ERK2/T∗EY∗). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.Display Omitted► Docking peptides induce local and long-range changes in p38a observed by solution NMR ► Docking peptides induce similar structures in active and inactive p38α ► The allosteric mechanism of peptide-induced changes involves water molecules

The Third Conformation of p38α MAP Kinase Observed in Phosphorylated p38α and in Solution by Radha Akella; Xiaoshan Min; Qiong Wu; Kevin H. Gardner; Elizabeth J. Goldsmith (pp. 1571-1578).
MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T∗GY∗/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T∗GY∗) and ERK2 (ERK2/T∗EY∗). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.Display Omitted► Docking peptides induce local and long-range changes in p38a observed by solution NMR ► Docking peptides induce similar structures in active and inactive p38α ► The allosteric mechanism of peptide-induced changes involves water molecules

In Vivo Assembly of an Archaeal Virus Studied with Whole-Cell Electron Cryotomography by Chi-yu Fu; Kang Wang; Lu Gan; Jason Lanman; Reza Khayat; Mark J. Young; Grant J. Jensen; Peter C. Doerschuk; John E. Johnson (pp. 1579-1586).
We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.► Follow the assembly and maturation of STIV in a life-like state in whole cells ► Reveal assembly pathway of an innermembrane containing virus in the cell ► Reveal viral arrays and viral genome-packaging related particle reorganization ► Characterize the 3D structure and the formation of viral infection induced pyramids

In Vivo Assembly of an Archaeal Virus Studied with Whole-Cell Electron Cryotomography by Chi-yu Fu; Kang Wang; Lu Gan; Jason Lanman; Reza Khayat; Mark J. Young; Grant J. Jensen; Peter C. Doerschuk; John E. Johnson (pp. 1579-1586).
We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.► Follow the assembly and maturation of STIV in a life-like state in whole cells ► Reveal assembly pathway of an innermembrane containing virus in the cell ► Reveal viral arrays and viral genome-packaging related particle reorganization ► Characterize the 3D structure and the formation of viral infection induced pyramids

Structural and Functional Analysis of Phosphothreonine-Dependent FHA Domain Interactions by Simon Pennell; Sarah Westcott; Miguel Ortiz-Lombardía; Dony Patel; Jiejin Li; Timothy J. Nott; Duaa Mohammed; Roger S. Buxton; Michael B. Yaffe; Chandra Verma; Stephen J. Smerdon (pp. 1587-1595).
FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.► Identification of high-affinity phosphopeptides for the Mycobacterium tuberculosis Rv0020c FHA domain ► Structures of free and phosphopeptide-bound forms ► Mutagenic analysis defines specificity determinants of the interaction ► Molecular dynamics reveals the origin of FHA domain discrimination against pSer-containing epitopes

Structural and Functional Analysis of Phosphothreonine-Dependent FHA Domain Interactions by Simon Pennell; Sarah Westcott; Miguel Ortiz-Lombardía; Dony Patel; Jiejin Li; Timothy J. Nott; Duaa Mohammed; Roger S. Buxton; Michael B. Yaffe; Chandra Verma; Stephen J. Smerdon (pp. 1587-1595).
FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.► Identification of high-affinity phosphopeptides for the Mycobacterium tuberculosis Rv0020c FHA domain ► Structures of free and phosphopeptide-bound forms ► Mutagenic analysis defines specificity determinants of the interaction ► Molecular dynamics reveals the origin of FHA domain discrimination against pSer-containing epitopes

Nanometer Propagation of Millisecond Motions in V-Type Allostery by James M. Lipchock; J. Patrick Loria (pp. 1596-1607).
Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, which is catalytically inactive for glutamine hydrolysis until the allosteric effector, N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binds 30 Å away. In the apo state, NMR relaxation dispersion experiments indicate the absence of millisecond (ms) timescale motions. Binding of the PRFAR to form the active ternary complex is endothermic with a large positive entropy change. In addition, there is a protein wide enhancement of conformational motions in the ternary complex, which connect the two active sites. NMR chemical shift changes and acrylamide quenching experiments suggest that little in the way of structural changes accompany these motions. The data indicate that enzyme activation in the ternary complex is primarily due to an enhancement of ms motions that allows formation of a population of enzymatically active conformers.► apo IGPS displays few millisecond motions ► Allosteric ligand binding is entropically dominated ► Protein-wide millisecond motions are enhanced upon allosteric ligand binding ► The flexible residues form a continuous network between active sites

Nanometer Propagation of Millisecond Motions in V-Type Allostery by James M. Lipchock; J. Patrick Loria (pp. 1596-1607).
Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, which is catalytically inactive for glutamine hydrolysis until the allosteric effector, N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binds 30 Å away. In the apo state, NMR relaxation dispersion experiments indicate the absence of millisecond (ms) timescale motions. Binding of the PRFAR to form the active ternary complex is endothermic with a large positive entropy change. In addition, there is a protein wide enhancement of conformational motions in the ternary complex, which connect the two active sites. NMR chemical shift changes and acrylamide quenching experiments suggest that little in the way of structural changes accompany these motions. The data indicate that enzyme activation in the ternary complex is primarily due to an enhancement of ms motions that allows formation of a population of enzymatically active conformers.► apo IGPS displays few millisecond motions ► Allosteric ligand binding is entropically dominated ► Protein-wide millisecond motions are enhanced upon allosteric ligand binding ► The flexible residues form a continuous network between active sites

Developing Three-Dimensional Models of Putative-Folding Intermediates of the HDV Ribozyme by Cédric Reymond; Dominique Lévesque; Martin Bisaillon; Jean-Pierre Perreault (pp. 1608-1616).
Both the role and the interacting partners of an RNA molecule can change depending on its tertiary structure. Consequently, it is important to be able to accurately predict the complete folding pathway of an RNA molecule. The hepatitis delta virus (HDV) ribozyme is a small catalytic RNA with the greatest number of folding intermediates making it the model of choice with which to address this problem. The tertiary structures of the known putative intermediates along the folding pathway of the HDV ribozyme were predicted using the Macromolecular Conformations Symbolic programming (MC-Sym) software. The structures obtained by this method received physical support from Selective 2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). The analysis of these structures elucidated several features of the HDV ribozyme. In addition, this report represents an application for MC-Sym that permits progression one step further toward the computer prediction of an RNA molecule-folding pathway.► Three-dimensional representation of the most complex folding pathway elucidated to date ► The analysis of these structures elucidated several features of the HDV ribozyme ► Progressing toward the computer prediction of an RNA molecule's folding pathway ► Novel way of learning from an RNA molecule's folding pathway

Developing Three-Dimensional Models of Putative-Folding Intermediates of the HDV Ribozyme by Cédric Reymond; Dominique Lévesque; Martin Bisaillon; Jean-Pierre Perreault (pp. 1608-1616).
Both the role and the interacting partners of an RNA molecule can change depending on its tertiary structure. Consequently, it is important to be able to accurately predict the complete folding pathway of an RNA molecule. The hepatitis delta virus (HDV) ribozyme is a small catalytic RNA with the greatest number of folding intermediates making it the model of choice with which to address this problem. The tertiary structures of the known putative intermediates along the folding pathway of the HDV ribozyme were predicted using the Macromolecular Conformations Symbolic programming (MC-Sym) software. The structures obtained by this method received physical support from Selective 2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). The analysis of these structures elucidated several features of the HDV ribozyme. In addition, this report represents an application for MC-Sym that permits progression one step further toward the computer prediction of an RNA molecule-folding pathway.► Three-dimensional representation of the most complex folding pathway elucidated to date ► The analysis of these structures elucidated several features of the HDV ribozyme ► Progressing toward the computer prediction of an RNA molecule's folding pathway ► Novel way of learning from an RNA molecule's folding pathway

Structural Basis for the Differential Effects of CaBP1 and Calmodulin on CaV1.2 Calcium-Dependent Inactivation by Felix Findeisen; Daniel L. Minor Jr. (pp. 1617-1631).
Calcium-binding protein 1 (CaBP1), a calmodulin (CaM) homolog, endows certain voltage-gated calcium channels (CaVs) with unusual properties. CaBP1 inhibits CaV1.2 calcium-dependent inactivation (CDI) and introduces calcium-dependent facilitation (CDF). Here, we show that the ability of CaBP1 to inhibit CaV1.2 CDI and induce CDF arises from interaction between the CaBP1 N-lobe and interlobe linker residue Glu94. Unlike CaM, where functional EF hands are essential for channel modulation, CDI inhibition does not require functional CaBP1 EF hands. Furthermore, CaBP1-mediated CDF has different molecular requirements than CaM-mediated CDF. Overall, the data show that CaBP1 comprises two structural modules having separate functions: similar to CaM, the CaBP1 C-lobe serves as a high-affinity anchor that binds the CaV1.2 IQ domain at a site that overlaps with the Ca2+/CaM C-lobe site, whereas the N-lobe/linker module houses the elements required for channel modulation. Discovery of this division provides the framework for understanding how CaBP1 regulates CaVs.► CaBP1 lobes have separable functions ► CaBP1 N-lobe-linker interactions are central to CaV modulation ► Ca2+/CaBP1 C-lobe and Ca2+/CaM C-lobe binding sites on CaV1.2 IQ domain overlap ► Functional EF hands are not essential for CaBP1 inhibition of CaV1.2 CDI

Structural Basis for the Differential Effects of CaBP1 and Calmodulin on CaV1.2 Calcium-Dependent Inactivation by Felix Findeisen; Daniel L. Minor Jr. (pp. 1617-1631).
Calcium-binding protein 1 (CaBP1), a calmodulin (CaM) homolog, endows certain voltage-gated calcium channels (CaVs) with unusual properties. CaBP1 inhibits CaV1.2 calcium-dependent inactivation (CDI) and introduces calcium-dependent facilitation (CDF). Here, we show that the ability of CaBP1 to inhibit CaV1.2 CDI and induce CDF arises from interaction between the CaBP1 N-lobe and interlobe linker residue Glu94. Unlike CaM, where functional EF hands are essential for channel modulation, CDI inhibition does not require functional CaBP1 EF hands. Furthermore, CaBP1-mediated CDF has different molecular requirements than CaM-mediated CDF. Overall, the data show that CaBP1 comprises two structural modules having separate functions: similar to CaM, the CaBP1 C-lobe serves as a high-affinity anchor that binds the CaV1.2 IQ domain at a site that overlaps with the Ca2+/CaM C-lobe site, whereas the N-lobe/linker module houses the elements required for channel modulation. Discovery of this division provides the framework for understanding how CaBP1 regulates CaVs.► CaBP1 lobes have separable functions ► CaBP1 N-lobe-linker interactions are central to CaV modulation ► Ca2+/CaBP1 C-lobe and Ca2+/CaM C-lobe binding sites on CaV1.2 IQ domain overlap ► Functional EF hands are not essential for CaBP1 inhibition of CaV1.2 CDI

Crystal Structure of HIV-1 Primary Receptor CD4 in Complex with a Potent Antiviral Antibody by Michael M. Freeman; Michael S. Seaman; Sophia Rits-Volloch; Xinguo Hong; Chia-Ying Kao; David D. Ho; Bing Chen (pp. 1632-1641).
Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 Å resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121–125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.► Crystal structure of HIV-1 primary receptor CD4 in complex with an antiviral antibody ► Molecular mechanism of HIV-1 entry

Crystal Structure of HIV-1 Primary Receptor CD4 in Complex with a Potent Antiviral Antibody by Michael M. Freeman; Michael S. Seaman; Sophia Rits-Volloch; Xinguo Hong; Chia-Ying Kao; David D. Ho; Bing Chen (pp. 1632-1641).
Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 Å resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121–125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.► Crystal structure of HIV-1 primary receptor CD4 in complex with an antiviral antibody ► Molecular mechanism of HIV-1 entry

Structural Characterization of the DAXX N-Terminal Helical Bundle Domain and Its Complex with Rassf1C by Eric Escobar-Cabrera; Desmond K.W. Lau; Serena Giovinazzi; Alexander M. Ishov; Lawrence P. McIntosh (pp. 1642-1653).
DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (KD ∼60 μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.Display Omitted► The modular structure of human DAXX was characterized ► DAXX contains a left-handed helical bundle, distinct from the Sin3 PAH domain ► Rassf1C undergoes a coil-to-helix transition upon binding the DAXX helical bundle ► A DAXX recognition motif contains hydrophobic and flanking acidic residues

Structural Characterization of the DAXX N-Terminal Helical Bundle Domain and Its Complex with Rassf1C by Eric Escobar-Cabrera; Desmond K.W. Lau; Serena Giovinazzi; Alexander M. Ishov; Lawrence P. McIntosh (pp. 1642-1653).
DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (KD ∼60 μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.Display Omitted► The modular structure of human DAXX was characterized ► DAXX contains a left-handed helical bundle, distinct from the Sin3 PAH domain ► Rassf1C undergoes a coil-to-helix transition upon binding the DAXX helical bundle ► A DAXX recognition motif contains hydrophobic and flanking acidic residues

Structural Diversity in Integrin/Talin Interactions by Nicholas J. Anthis; Kate L. Wegener; David R. Critchley; Iain D. Campbell (pp. 1654-1666).
The adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the β-integrin subunit. Structural studies of this interaction have hitherto largely focused on the β3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between β1A, β1D, and β3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/β tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/β1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the β tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.Display Omitted► Integrin/talin interactions vary in biologically significant ways between isoforms ► The high affinity of the muscle-specific integrin β1D/Talin2 complex is explained ► NMR suggests a role for conformational entropy in integrin tail recognition ► Targeted mutation of integrin tail increases the affinity for talin 1000-fold

Structural Diversity in Integrin/Talin Interactions by Nicholas J. Anthis; Kate L. Wegener; David R. Critchley; Iain D. Campbell (pp. 1654-1666).
The adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the β-integrin subunit. Structural studies of this interaction have hitherto largely focused on the β3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between β1A, β1D, and β3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/β tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/β1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the β tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.Display Omitted► Integrin/talin interactions vary in biologically significant ways between isoforms ► The high affinity of the muscle-specific integrin β1D/Talin2 complex is explained ► NMR suggests a role for conformational entropy in integrin tail recognition ► Targeted mutation of integrin tail increases the affinity for talin 1000-fold

Allosteric Activation Mechanism of the Mycobacterium tuberculosis Receptor Ser/Thr Protein Kinase, PknB by T. Noelle Lombana; Nathaniel Echols; Matthew C. Good; Nathan D. Thomsen; Ho-Leung Ng; Andrew E. Greenstein; Arnold M. Falick; David S. King; Tom Alber (pp. 1667-1677).
The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate >30 Å from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how “back-to-back” N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.► Dimerization allosterically activates M. tuberculosis Ser/Thr protein kinase PknB ► N-lobe dimer interface mutations block PknB-dependent phosphorylation in vivo ► N-lobe interface mutants adopt multiple, monomeric, inactive conformations ► N-lobe pairing affords a general mechanism of activation for homologous kinases

Allosteric Activation Mechanism of the Mycobacterium tuberculosis Receptor Ser/Thr Protein Kinase, PknB by T. Noelle Lombana; Nathaniel Echols; Matthew C. Good; Nathan D. Thomsen; Ho-Leung Ng; Andrew E. Greenstein; Arnold M. Falick; David S. King; Tom Alber (pp. 1667-1677).
The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate >30 Å from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how “back-to-back” N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.► Dimerization allosterically activates M. tuberculosis Ser/Thr protein kinase PknB ► N-lobe dimer interface mutations block PknB-dependent phosphorylation in vivo ► N-lobe interface mutants adopt multiple, monomeric, inactive conformations ► N-lobe pairing affords a general mechanism of activation for homologous kinases

Toward a Structure Determination Method for Biomineral-Associated Protein Using Combined Solid- State NMR and Computational Structure Prediction by David L. Masica; Jason T. Ash; Moise Ndao; Gary P. Drobny; Jeffrey J. Gray (pp. 1678-1687).
Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized.Display Omitted► Novel method for determining the structure of protein-absorbed states ► Method combines solid-state NMR and computational structure prediction ► Partial high-resolution structure of human salivary statherin binding tooth enamel ► Statherin binds hydroxyapatite via a highly charged helical-binding domain

Toward a Structure Determination Method for Biomineral-Associated Protein Using Combined Solid- State NMR and Computational Structure Prediction by David L. Masica; Jason T. Ash; Moise Ndao; Gary P. Drobny; Jeffrey J. Gray (pp. 1678-1687).
Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized.Display Omitted► Novel method for determining the structure of protein-absorbed states ► Method combines solid-state NMR and computational structure prediction ► Partial high-resolution structure of human salivary statherin binding tooth enamel ► Statherin binds hydroxyapatite via a highly charged helical-binding domain
The Human Breast Cancer Resistance Protein (BCRP/ABCG2) Shows Conformational Changes with Mitoxantrone by Mark F. Rosenberg; Zsolt Bikadi; Janice Chan; Xiaoping Liu; Zhanglin Ni; Xiaokun Cai; Robert C. Ford; Qingcheng Mao (pp. 1688-1689).
Structure of the PTEN-like Region of Auxilin, a Detector of Clathrin-Coated Vesicle Budding by Rong Guan; Han Dai; Stephen C. Harrison; Tomas Kirchhausen (pp. 1688-1688).
The Human Breast Cancer Resistance Protein (BCRP/ABCG2) Shows Conformational Changes with Mitoxantrone by Mark F. Rosenberg; Zsolt Bikadi; Janice Chan; Xiaoping Liu; Zhanglin Ni; Xiaokun Cai; Robert C. Ford; Qingcheng Mao (pp. 1688-1689).
Structure of the PTEN-like Region of Auxilin, a Detector of Clathrin-Coated Vesicle Budding by Rong Guan; Han Dai; Stephen C. Harrison; Tomas Kirchhausen (pp. 1688-1688).
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