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Structure (v.19, #7)

In This Issue (pp. v-vi).

A New Piece in the 3D Jigsaw of Malaria Parasite by Mariusz Jaskolski (pp. 901-902).
The structural insights presented by Hansen et al. in this issue of Structure on how a key malaria parasite protease is blocked by its inhibitor may provide a flicker of hope in the desperate struggle to develop drugs against one of the most severe health problems.

The Endless Subtleties of RNA-Protein Complexes by Eric Westhof; Valérie Fritsch (pp. 902-903).
New NMR structures of complexes between double-strand RNA binding domains and their RNA hairpin substrates reported by Wang et al. in this issue of Structure reveal striking adaptations in both the protein recognition element and in the RNA.

Pass the Jelly Rolls by Reza Khayat; John E. Johnson (pp. 904-906).
The crystal structure of the vaccinia virus D13 protein presented by Bahar et al. in this issue of Structure displays fused “virus jelly roll” folds, ubiquitous among dsDNA icosahedral viruses. Although D13 is not present in the mature virus, its structure suggests its evolutionary descent from an ancient icosahedral ancestor.

Dynamic Allostery: Linkers Are Not Merely Flexible by Buyong Ma; Chung-Jung Tsai; Türkan Haliloğlu; Ruth Nussinov (pp. 907-917).
Most proteins consist of multiple domains. How do linkers efficiently transfer information between sites that are on different domains to activate the protein? Mere flexibility only implies that the conformations would be sampled. For fast timescales between triggering events and cellular response, which often involves large conformational change, flexibility on its own may not constitute a good solution. We posit that successive conformational states along major allosteric propagation pathways are pre-encoded in linker sequences where each state is encoded by the previous one. The barriers between these states that are hierarchically populated are lower, achieving faster timescales even for large conformational changes. We further propose that evolution has optimized the linker sequences and lengths for efficiency, which explains why mutations in linkers may affect protein function and review the literature in this light.

Structural Basis for the Regulation of Cysteine-Protease Activity by a New Class of Protease Inhibitors in Plasmodium by Guido Hansen; Anna Heitmann; Tina Witt; Honglin Li; Hualiang Jiang; Xu Shen; Volker T. Heussler; Annika Rennenberg; Rolf Hilgenfeld (pp. 919-929).
Plasmodium cysteine proteases are essential for host-cell invasion and egress, hemoglobin degradation, and intracellular development of the parasite. The temporal, site-specific regulation of cysteine-protease activity is a prerequisite for survival and propagation of Plasmodium. Recently, a new family of inhibitors of cysteine proteases (ICPs) with homologs in at least eight Plasmodium species has been identified. Here, we report the 2.6 Å X-ray crystal structure of the C-terminal, inhibitory domain of ICP from P. berghei (PbICP-C) in a 1:1 complex with falcipain-2, an important hemoglobinase of Plasmodium. The structure establishes Plasmodium ICP as a member of the I42 class of chagasin-like protease inhibitors but with large insertions and differences in the binding mode relative to other family members. Furthermore, the PbICP-C structure explains why host-cell cathepsin B-like proteases and, most likely, also the protease-like domain of Plasmodium SERA5 (serine-repeat antigen 5) are no targets for ICP.► Structure of ICP-C from P. berghei in complex with falcipain-2 ► The inhibitor interacts with the protease via four loops ► Mutagenic analysis defines specificity determinants of the interaction ► The C-terminal domain of Plasmodium ICP is sufficient for full protease inhibition

Protein-RNA and Protein-Protein Recognition by Dual KH1/2 Domains of the Neuronal Splicing Factor Nova-1 by Marianna Teplova; Lucy Malinina; Jennifer C. Darnell; Jikui Song; Min Lu; Ruben Abagyan; Kiran Musunuru; Alexei Teplov; Stephen K. Burley; Robert B. Darnell; Dinshaw J. Patel (pp. 930-944).
Nova onconeural antigens are neuron-specific RNA-binding proteins implicated in paraneoplastic opsoclonus-myoclonus-ataxia (POMA) syndrome. Nova harbors three K-homology (KH) motifs implicated in alternate splicing regulation of genes involved in inhibitory synaptic transmission. We report the crystal structure of the first two KH domains (KH1/2) of Nova-1 bound to an in vitro selected RNA hairpin, containing a UCAG-UCAC high-affinity binding site. Sequence-specific intermolecular contacts in the complex involve KH1 and the second UCAC repeat, with the RNA scaffold buttressed by interactions between repeats. Whereas the canonical RNA-binding surface of KH2 in the above complex engages in protein-protein interactions in the crystalline state, the individual KH2 domain can sequence-specifically target the UCAC RNA element in solution. The observed antiparallel alignment of KH1 and KH2 domains in the crystal structure of the complex generates a scaffold that could facilitate target pre-mRNA looping on Nova binding, thereby potentially explaining Nova's functional role in splicing regulation.Display Omitted4Crystal structure of Nova-1 tandem KH1/2 domains bound to UCAN repeat RNA hairpin 4KH1 domain targets UCAG-UCAC tandem tetranucleotide sites in hairpin loop 4KH2 domain targets UCAY repeat single-stranded RNAs in a sequence specific manner 4KH1/KH2 pseudodimer induces target pre-mRNA looping on Nova binding

NusA Interaction with the α Subunit of E. coli RNA Polymerase Is via the UP Element Site and Releases Autoinhibition by Kristian Schweimer; Stefan Prasch; Pagadala Santhanam Sujatha; Mikhail Bubunenko; Max E. Gottesman; Paul Rösch (pp. 945-954).
Elongating Escherichia coli RNAP is modulated by NusA protein. The C-terminal domain (CTD) of the RNAP α subunit (αCTD) interacts with the acidic CTD 2 (AR2) of NusA, releasing the autoinhibitory blockade of the NusA S1-KH1-KH2 motif and allowing NusA to bind nascent nut spacer RNA. We determined the solution conformation of the AR2:αCTD complex. The αCTD residues that interface with AR2 are identical to those that recognize UP promoter elements A nusA-ΔAR2 mutation does not affect UP-dependent rrnH transcription initiation in vivo. Instead, the mutation inhibits Rho-dependent transcription termination at phage λ tR1, which lies adjacent to the λ nutR sequence. The Rho-dependent λ timm terminator, which is not preceded by a λ nut sequence, is fully functional. We propose that constitutive binding of NusA-ΔAR2 to λ nutR occludes Rho. In addition, the mutation confers a dominant defect in exiting stationary phase.Display Omitted► Solution structure of AR2:αCTD complex shows asymmetric interaction of two (HhH)2 domains ► NusA autoinhibition is caused by interaction between AR2 and KH domains, preventing RNA binding ► nut spacer RNA binds to KH1 domain of NusA ► NusA deleted for AR2 competes in vivo with Rho termination factor at λ nutR tR1

Protein-Protein Complex Structure Predictions by Multimeric Threading and Template Recombination by Srayanta Mukherjee; Yang Zhang (pp. 955-966).
The total number of protein-protein complex structures currently available in the Protein Data Bank (PDB) is six times smaller than the total number of tertiary structures in the PDB, which limits the power of homology-based approaches to complex structure modeling. We present a threading-recombination approach, COTH, to boost the protein complex structure library by combining tertiary structure templates with complex alignments. The query sequences are first aligned to complex templates using a modified dynamic programming algorithm, guided by ab initio binding-site predictions. The monomer alignments are then shifted to the multimeric template framework by structural alignments. COTH was tested on 500 nonhomologous dimeric proteins, which can successfully detect correct templates for 50% of the cases after homologous templates are excluded, which significantly outperforms conventional homology modeling algorithms. It also shows a higher accuracy in interface modeling than rigid-body docking of unbound structures from ZDOCK although with lower coverage. These data demonstrate new avenues to model complex structures from nonhomologous templates.Display Omitted► A novel algorithm for template-based protein-protein complex structure prediction ► Significant ability to recognize nonhomologous complex templates ► Boost protein quaternary structure library by tertiary structure recombination ► Complementary to rigid-body protein-protein docking methods

Allostery in Protein Domains Reflects a Balance of Steric and Hydrophobic Effects by Jeremy L. England (pp. 967-975).
Allosteric conformational change underlies biological function in many proteins. Allostery refers to a conformational event in which one region of a protein undergoes structural rearrangement in response to a stimulus applied to a different region of the same protein. Here, I show for a variety of proteins that a simple, phenomenological model of the dependence of protein conformation on hydrophobic burial energy allows one to compute low-energy conformational fluctuations for a given sequence by using linear programming to find optimized combinations of sequence-specific hydrophobic burial modes that satisfy steric constraints. From these fluctuations one may calculate allosteric couplings between different sites in a protein domain. Although the physical basis of protein structure is complex and multifactorial, a simplified description of conformational energy in terms of the hydrophobic effect alone is sufficient to give a mechanistic explanation for many biologically important allosteric events.► Every protein sequence has a corresponding hierarchy of hydrophobic burial modes ► Native structure arises from a sterically constrained competition among burial modes ► Near-native structural fluctuations provide the basis for allostery in many cases ► Many allosteric events result from low-energy seesawing between pairs of modes

Fragment-Based Phase Extension for Three-Dimensional Structure Determination of Membrane Proteins by Electron Crystallography by Goragot Wisedchaisri; Tamir Gonen (pp. 976-987).
In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.► Phase extension approach was developed to solve membrane protein structures ► A new approach was developed to accelerate structure analysis by electron crystallography

Interconversion of Two GDP-Bound Conformations and Their Selection in an Arf-Family Small G Protein by Hideyasu Okamura; Masaki Nishikiori; Hongyu Xiang; Masayuki Ishikawa; Etsuko Katoh (pp. 988-998).
ADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotideMg2+-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg2+ has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg2+, respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram.Display Omitted► The Arl8 core domain reversibly transitions between two GDP-bound conformations ► The N-terminal helix selects for the typical Arf-family GDP form in Arl8-GDP complex ► Mg2+ binds to a GTP-like form in Arl8-GDP in a population-shift manner ► The thermodynamics of the interswitch toggle event in Arl8 have been characterized

Structure of a Yeast RNase III dsRBD Complex with a Noncanonical RNA Substrate Provides New Insights into Binding Specificity of dsRBDs by Zhonghua Wang; Elon Hartman; Kevin Roy; Guillaume Chanfreau; Juli Feigon (pp. 999-1010).
dsRBDs often bind dsRNAs with some specificity, yet the basis for this is poorly understood. Rnt1p, the major RNase III in Saccharomyces cerevisiae, cleaves RNA substrates containing hairpins capped by A/uGNN tetraloops, using its dsRBD to recognize a conserved tetraloop fold. However, the identification of a Rnt1p substrate with an AAGU tetraloop raised the question of whether Rnt1p binds to this noncanonical substrate differently than to A/uGNN tetraloops. The solution structure of Rnt1p dsRBD bound to an AAGU-capped hairpin reveals that the tetraloop undergoes a structural rearrangement upon binding to Rnt1p dsRBD to adopt a backbone conformation that is essentially the same as the AGAA tetraloop, and indicates that a conserved recognition mode is used for all Rnt1p substrates. Comparison of free and RNA-bound Rnt1p dsRBD reveals that tetraloop-specific binding requires a conformational change in helix α1. Our findings provide a unified model of binding site selection by this dsRBD.Display Omitted► AAGU tetraloop undergoes a structural rearrangement upon Rnt1p dsRBD binding ► AAGU tetraloop has a canonical A/uGNN tetraloop fold in complex with Rnt1p dsRBD ► Rnt1p dsRBD helix α1 undergoes a conformational change upon RNA tetraloop binding ► Rnt1p has a conserved recognition mode for all Rnt1p substrates

Insights into the Evolution of a Complex Virus from the Crystal Structure of Vaccinia Virus D13 by Mohammad W. Bahar; Stephen C. Graham; David I. Stuart; Jonathan M. Grimes (pp. 1011-1020).
The morphogenesis of poxviruses such as vaccinia virus (VACV) sees the virion shape mature from spherical to brick-shaped. Trimeric capsomers of the VACV D13 protein form a transitory, stabilizing lattice on the surface of the initial spherical immature virus particle. The crystal structure of D13 reveals that this major scaffolding protein comprises a double β barrel “jelly-roll” subunit arranged as pseudo-hexagonal trimers. These structural features are characteristic of the major capsid proteins of a lineage of large icosahedral double-stranded DNA viruses including human adenovirus and the bacteriophages PRD1 and PM2. Structure-based phylogenetic analysis confirms that VACV belongs to this lineage, suggesting that (analogously to higher organism embryogenesis) early poxvirus morphogenesis reflects their evolution from a lineage of viruses sharing a common icosahedral ancestor.► Poxvirus D13 acts as a scaffold for the morphogenesis of spherical immature virions ► D13 has a double “jelly-roll” structure, like other large DNA virus capsid proteins ► Structure-based phylogenetics places D13 into an icosahedral viral lineage ► Poxvirus morphogenesis reflects Vaccinia virus evolution from an icosahedral ancestor

The Structure of E. coli IgG-Binding Protein D Suggests a General Model for Bending and Binding in Trimeric Autotransporter Adhesins by Jack C. Leo; Andrzej Lyskowski; Katarina Hattula; Marcus D. Hartmann; Heinz Schwarz; Sarah J. Butcher; Dirk Linke; Andrei N. Lupas; Adrian Goldman (pp. 1021-1030).
The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells.Display Omitted► The first TAA structure containing both a head domain and a complete stalk ► A β-minidomain mediates the change from right-handed to left-handed supercoiling ► The binding sites for IgG and IgA are in the stalk ► Large cavities in the stalk may explain how bending of TAAs occurs
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