Structure (v.19, #12)

In This Issue (vii-ix).

Face-to-Face, Pak-to-Pak by Kimberly A. Malecka; Jeffrey R. Peterson (1723-1724).
Biochemical and structural studies of p21-activated kinase 1 (Pak1) by Wang and colleagues in this issue of Structure reveal the structural basis for Pak1 trans-autophosphorylation of the activation loop, a critical step in the activation of kinases.

Progress at Last by Ron Elber (1725).
Refinement of protein structures from a correct topology to atomically detailed resolution has proven remarkably difficult. Jian et al. (in this issue of Structure) illustrate a significant advance in this task by carefully incorporating into the refinement process many body interactions extracted from fragment statistics.

Phosphorylation at the Interface by Fred P. Davis (1726-1727).
Proteomic studies have identified thousands of eukaryotic phosphorylation sites (phosphosites), but few are functionally characterized. Nishi et al., in this issue of Structure, characterize phosphosites at protein-protein interfaces and estimate the effect of their phosphorylation on interaction affinity, by combining proteomics data with protein structures.

Creating Order out of Disorder: Structural Imprint of GAPDH on CP12 by Brigitte Gontero; Luisana Avilan (1728-1729).
The work presented by Matsumura et al. in this issue of Structure describes the structure of the ternary GAPDH-NAD-CP12 and the binary NAD-GAPDH complex in the cyanobacterium Synechococcus elongatus.

Terpenoids are a diverse group of natural products with a myriad functions in ecological interactions of living organisms. Terpenoid synthases provide a unique reservoir of enzymes for the metabolic engineering of advanced biofuels and high-value bioproducts (McAndrew et al. in this issue of Structure).

First Crystal Structures of Na+,K+-ATPase: New Light on the Oldest Ion Pump by Chikashi Toyoshima; Ryuta Kanai; Flemming Cornelius (1732-1738).
Na+,K+-adenosine triphosphatase (NKA) is the first P-type ion translocating adenosine triphosphatase (ATPase) ever identified, and the significance of this class of proteins was highlighted by the 1997 Nobel Prize in Chemistry awarded to Jens C. Skou for the discovery in 1957. More than half a century passed between the initial identification and the publication of a high-resolution crystal structure of NKA. Although the new crystal structures provided many surprises and insights, structural biology on this system remains challenging, as NKA is a very difficult protein to crystallize. Here we explain the reasons behind the challenges, introduce a mechanism that governs the function, and summarize current knowledge of NKA structure in comparison with another member of the P-type ATPase family, Ca2+-ATPase.

Real Space Refinement of Crystal Structures with Canonical Distributions of Electrons by Simon W. Ginzinger; Markus Gruber; Hans Brandstetter; Manfred J. Sippl (1739-1743).
Recurring groups of atoms in molecules are surrounded by specific canonical distributions of electrons. Deviations from these distributions reveal unrealistic molecular geometries. Here, we show how canonical electron densities can be combined with classical electron densities derived from X-ray diffraction experiments to drive the real space refinement of crystal structures. The refinement process generally yields superior molecular models with reduced excess electron densities and improved stereochemistry without compromising the agreement between molecular models and experimental data.► Recurring groups of atoms in proteins are surrounded by canonical electron densities ► Deviations from canonical densities reveal unrealistic molecular geometries ► Canonical density refinement removes electron excess and improves stereochemistry

Automated Prediction of Protein Association Rate Constants by Sanbo Qin; Xiaodong Pang; Huan-Xiang Zhou (1744-1751).
Display Omitted► A method is presented for automated prediction of protein association rates ► The prediction method is both accurate and robust, and has wide applications ► With this method, half of the protein association problem is now solved

p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative “substrate” is projected into the active site of the “enzyme.” The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.► Unphosphorylated PAK-KD dynamically adopts both active and inactive conformations ► Unphosphorylated PAK-KD forms an authentic enzyme-substrate complex

Extracellular Complexes of the Hematopoietic Human and Mouse CSF-1 Receptor Are Driven by Common Assembly Principles by Jonathan Elegheert; Ambroise Desfosses; Alexander V. Shkumatov; Xiongwu Wu; Nathalie Bracke; Kenneth Verstraete; Kathleen Van Craenenbroeck; Bernard R. Brooks; Dmitri I. Svergun; Bjorn Vergauwen; Irina Gutsche; Savvas N. Savvides (1762-1772).
The hematopoietic colony stimulating factor-1 receptor (CSF-1R or FMS) is essential for the cellular repertoire of the mammalian immune system. Here, we report a structural and mechanistic consensus for the assembly of human and mouse CSF-1:CSF-1R complexes. The EM structure of the complete extracellular assembly of the human CSF-1:CSF-1R complex reveals how receptor dimerization by CSF-1 invokes a ternary complex featuring extensive homotypic receptor contacts and striking structural plasticity at the extremities of the complex. Studies by small-angle X-ray scattering of unliganded hCSF-1R point to large domain rearrangements upon CSF-1 binding, and provide structural evidence for the relevance of receptor predimerization at the cell surface. Comparative structural and binding studies aiming to dissect the assembly principles of human and mouse CSF-1R complexes, including a quantification of the CSF-1/CSF-1R species cross-reactivity, show that bivalent cytokine binding to receptor coupled to ensuing receptor-receptor interactions are common denominators in extracellular complex formation.Display Omitted► Assembly of extracellular human and mouse CSF-1:CSF-1R complexes ► Thermodynamics of complex formation and characterization of species cross-reactivity ► Bivalent binding of CSF-1 to CSF-1R ectodomains underlies complex formation ► A mechanistic consensus for human and mouse CSF-1:CSF-1R complexes

Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites by Svetlana Petkun; Rong Shi; Yunge Li; Abdalin Asinas; Christine Munger; Linhua Zhang; Mandy Waclawek; Basem Soboh; R. Gary Sawers; Miroslaw Cygler (1773-1783).
[NiFe]-hydrogenases are multimeric proteins. The large subunit contains the NiFe(CN)2CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)2CO active center requires six Hyp accessory proteins. The synthesis of the CN ligands is catalyzed by the combined actions of HypF and HypE using carbamoylphosphate as a substrate. We report the structure of Escherichia coli HypF(92–750) lacking the N-terminal acylphosphatase domain. HypF(92–750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn2+ ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14 Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.

One of critical difficulties of molecular dynamics (MD) simulations in protein structure refinement is that the physics-based energy landscape lacks a middle-range funnel to guide nonnative conformations toward near-native states. We propose to use the target model as a probe to identify fragmental analogs from PDB. The distance maps are then used to reshape the MD energy funnel. The protocol was tested on 181 benchmarking and 26 CASP targets. It was found that structure models of correct folds with TM-score >0.5 can be often pulled closer to native with higher GDT-HA score, but improvement for the models of incorrect folds (TM-score <0.5) are much less pronounced. These data indicate that template-based fragmental distance maps essentially reshaped the MD energy landscape from golf-course-like to funnel-like ones in the successfully refined targets with a radius of TM-score ∼0.5. These results demonstrate a new avenue to improve high-resolution structures by combining knowledge-based template information with physics-based MD simulations.Display Omitted► Development of a protocol for atomic-level protein structure refinement ► Reshaping physical energy landscape by fragmental templates ► Consistent structural refinement in both large-scale benchmark and blind tests ► Combination of knowledge-based information with physics-based MD simulations

Galline Ex-FABP Is an Antibacterial Siderocalin and a Lysophosphatidic Acid Sensor Functioning through Dual Ligand Specificities by Colin Correnti; Matthew C. Clifton; Rebecca J. Abergel; Ben Allred; Trisha M. Hoette; Mario Ruiz; Ranieri Cancedda; Kenneth N. Raymond; Fiorella Descalzi; Roland K. Strong (1796-1806).
Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8 Å crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding copurified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.► Ex-FABP is bacteriostatic through iron sequestration by siderophore binding ► Ex-FABP binds an extended range of siderophores versus Siderocalin ► The extended Ex-FABP calyx defines a second binding site for lysophosphatidic acid

Phosphorylation in Protein-Protein Binding: Effect on Stability and Function by Hafumi Nishi; Kosuke Hashimoto; Anna R. Panchenko (1807-1815).
Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. Analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and that binding hotspots tend to be phosphorylated in heterooligomers. Although the majority of complexes do not show significant estimated stability differences upon phosphorylation or dephosphorylation, for about one-third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are more likely to be evolutionary conserved than other interfacial residues.Display Omitted► Phosphosites tend to be located on interfaces in protein structural complexes ► Phosphorylation does not change binding affinity significantly for more than half cases ► Phosphorylation at binding hotspots is frequent and may disrupt the complex formation ► Phosphosites are more conserved than other interface sites

Crystallographic Analysis of Polypyrimidine Tract-Binding Protein-Raver1 Interactions Involved in Regulation of Alternative Splicing by Amar Joshi; Miguel B. Coelho; Olga Kotik-Kogan; Peter J. Simpson; Stephen J. Matthews; Christopher W.J. Smith; Stephen Curry (1816-1825).
The polypyrimidine tract-binding protein (PTB) is an important regulator of alternative splicing. PTB-regulated splicing of α-tropomyosin is enhanced by Raver1, a protein with four PTB-Raver1 interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. We present the crystal structures of RRM2 in complex with PRI3 and PRI4 from Raver1, which—along with structure-based mutagenesis—reveal the molecular basis of their differential binding. High-affinity binding by Raver1 PRI3 involves shape-matched apolar contacts complemented by specific hydrogen bonds, a new variant of an established mode of peptide-RRM interaction. Our results refine the sequence of the PRI motif and place important structural constraints on functional models of PTB-Raver1 interactions. Our analysis indicates that the observed Raver1-PTB interaction is a general mode of binding that applies to Raver1 complexes with PTB paralogues such as nPTB and to complexes of Raver2 with PTB.► First crystal structures of PTB reveals how it binds Raver1 via PRI motifs ► Observed mode of binding is general for PRIs and PTB paralogues ► Results place constraints on models of splicing repression by PTB-Raver1 complexes

Tandem SAM Domain Structure of Human Caskin1: A Presynaptic, Self-Assembling Scaffold for CASK by Ryan L. Stafford; Elizabeth Hinde; Mary Jane Knight; Mario A. Pennella; Jason Ear; Michelle A. Digman; Enrico Gratton; James U. Bowie (1826-1836).
The synaptic scaffolding proteins CASK and Caskin1 are part of the fibrous mesh of proteins that organize the active zones of neural synapses. CASK binds to a region of Caskin1 called the CASK interaction domain (CID). Adjacent to the CID, Caskin1 contains two tandem sterile α motif (SAM) domains. Many SAM domains form polymers so they are good candidates for forming the fibrous structures seen in the active zone. We show here that the SAM domains of Caskin1 form a new type of SAM helical polymer. The Caskin1 polymer interface exhibits a remarkable segregation of charged residues, resulting in a high sensitivity to ionic strength in vitro. The Caskin1 polymers can be decorated with CASK proteins, illustrating how these proteins may work together to organize the cytomatrix in active zones.Display Omitted► The tandem SAM domains of Caskin1 assemble into a polymeric scaffold that binds CASK ► The Caskin1 SAM polymer interface is highly charged and sensitive to ionic strength ► Caskin1 may be part of the fibrous protein network in the synaptic active zone

Structural and Dynamic Determinants of Protein-Peptide Recognition by Onur Dagliyan; Elizabeth A. Proctor; Kevin M. D'Auria; Feng Ding; Nikolay V. Dokholyan (1837-1845).
Protein-peptide interactions play important roles in many cellular processes, including signal transduction, trafficking, and immune recognition. Protein conformational changes upon binding, an ill-defined peptide binding surface, and the large number of peptide degrees of freedom make the prediction of protein-peptide interactions particularly challenging. To address these challenges, we perform rapid molecular dynamics simulations in order to examine the energetic and dynamic aspects of protein-peptide binding. We find that, in most cases, we recapitulate the native binding sites and native-like poses of protein-peptide complexes. Inclusion of electrostatic interactions in simulations significantly improves the prediction accuracy. Our results also highlight the importance of protein conformational flexibility, especially side-chain movement, which allows the peptide to optimize its conformation. Our findings not only demonstrate the importance of sufficient sampling of the protein and peptide conformations, but also reveal the possible effects of electrostatics and conformational flexibility on peptide recognition.► Direct observation of protein-peptide binding in molecular dynamics simulations ► Electrostatic interactions guide protein-peptide recognition ► Direct observation of induced-fit phenomenon in peptides ► Novel method for protein-peptide docking

Structure Basis for the Regulation of Glyceraldehyde-3-Phosphate Dehydrogenase Activity via the Intrinsically Disordered Protein CP12 by Hiroyoshi Matsumura; Akihiro Kai; Takayuki Maeda; Masahiro Tamoi; Atsuko Satoh; Haruka Tamura; Mika Hirose; Taketo Ogawa; Natsuko Kizu; Akira Wadano; Tsuyoshi Inoue; Shigeru Shigeoka (1846-1854).
The reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.► The C-terminal region of CP12 is bound to GAPDH in complex with NAD ► The CP12 covers the substrate binding site of GAPDH ► NAD stabilizes the GAPDH-CP12 complex, but NADP does not ► An unexpected increase in negative charge potential emerges upon CP12 binding.

The Polydispersity of αB-Crystallin Is Rationalized by an Interconverting Polyhedral Architecture by Andrew J. Baldwin; Hadi Lioe; Gillian R. Hilton; Lindsay A. Baker; John L. Rubinstein; Lewis E. Kay; Justin L.P. Benesch (1855-1863).
We report structural models for the most abundant oligomers populated by the polydisperse molecular chaperone αB-crystallin. Subunit connectivity is determined by using restraints obtained from nuclear magnetic resonance spectroscopy and mass spectrometry measurements, enabling the construction of various oligomeric models. These candidate structures are filtered according to their correspondence with ion-mobility spectrometry data and cross-validated by using electron microscopy. The ensuing best-fit structures reveal the polyhedral architecture of αB-crystallin oligomers, and provide a rationale for their polydispersity and facile interconversion.► Hybrid study to determine structures of oligomers comprising a polydisperse ensemble ► Combined mass spectrometry (MS) and NMR approach to establish intersubunit connectivity ► Ion mobility MS and tandem-MS strategy to extract sizes of αB-crystallin oligomers ► Polyhedral architecture of oligomers rationalizes their facile interconversion

Structure Analysis of the IL-5 Ligand-Receptor Complex Reveals a Wrench-like Architecture for IL-5Rα by Edwin Patino; Alexander Kotzsch; Stefan Saremba; Joachim Nickel; Werner Schmitz; Walter Sebald; Thomas D. Mueller (1864-1875).
Interleukin-5 (IL-5) is the key mediator for the function of eosinophil granulocytes, whose deregulation is characteristic of hypereosinophilic diseases and presumably contributes to allergic asthma. IL-5 signaling involves two transmembrane receptors, IL-5Rα and the common β chain, which upon formation of the ternary complex activate the JAK/STAT signaling cascade. To investigate the mechanism underlying ligand-receptor recognition, we determined the structure of IL-5 bound to the extracellular domain of IL-5Rα. IL-5 makes contact with all three fibronectin III-like domains of IL-5Rα, with the receptor architecture resembling a wrench. Mutagenesis data provide evidence that this wrench-like architecture is likely preformed. The structure demonstrates that for steric reasons, homodimeric IL-5 can bind only one receptor molecule, even though two equivalent receptor-binding sites exist. In regard to strong efforts being made to develop IL-5 antagonists for treating asthma and hypereosinophilic diseases, the advances in molecular understanding provided by this structure are of greatest value.Display Omitted► Structure of the IL-5:IL-5Rα complex reveals a new receptor architecture for IL-5Rα ► Wrench-like architecture of the IL-5Rα ectodomain is shared with IL-13 receptors ► 1:1 stoichiometry of the binding of homodimeric IL-5 to IL-5Rα due to steric reasons ► IL-5Rα FNIII domain 1 is essential for high-affinity binding of IL-5

Structure of a Three-Domain Sesquiterpene Synthase: A Prospective Target for Advanced Biofuels Production by Ryan P. McAndrew; Pamela P. Peralta-Yahya; Andy DeGiovanni; Jose H. Pereira; Masood Z. Hadi; Jay D. Keasling; Paul D. Adams (1876-1884).
The sesquiterpene bisabolene was recently identified as a biosynthetic precursor to bisabolane, an advanced biofuel with physicochemical properties similar to those of D2 diesel. High-titer microbial bisabolene production was achieved using Abies grandis α-bisabolene synthase (AgBIS). Here, we report the structure of AgBIS, a three-domain plant sesquiterpene synthase, crystallized in its apo form and bound to five different inhibitors. Structural and biochemical characterization of the AgBIS terpene synthase Class I active site leads us to propose a catalytic mechanism for the cyclization of farnesyl diphosphate into bisabolene via a bisabolyl cation intermediate. Further, we describe the nonfunctional AgBIS Class II active site whose high similarity to bifunctional diterpene synthases makes it an important link in understanding terpene synthase evolution. Practically, the AgBIS crystal structure is important in future protein engineering efforts to increase the microbial production of bisabolene.► AgBIS is the first structure of a three-domain sesquiterpene synthase ► The structure was obtained in the apo form and bound to 5 different inhibitors ► A mechanism is proposed for the conversion of farnesyl diphosphate to bisabolene ► Insights are gained for the future engineering of this biofuels target

Structural Conservation of the Myoviridae Phage Tail Sheath Protein Fold by Anastasia A. Aksyuk; Lidia P. Kurochkina; Andrei Fokine; Farhad Forouhar; Vadim V. Mesyanzhinov; Liang Tong; Michael G. Rossmann (1885-1894).
Bacteriophage phiKZ is a giant phage that infects Pseudomonas aeruginosa, a human pathogen. The phiKZ virion consists of a 1450 Å diameter icosahedral head and a 2000 Å-long contractile tail. The structure of the whole virus was previously reported, showing that its tail organization in the extended state is similar to the well-studied Myovirus bacteriophage T4 tail. The crystal structure of a tail sheath protein fragment of phiKZ was determined to 2.4 Å resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3 Å resolution. Despite low sequence identity between these proteins, all of these structures have a similar fold. The crystal structure of the phiKZ tail sheath protein has been fitted into cryo-electron-microscopy reconstructions of the extended tail sheath and of a polysheath. The structural rearrangement of the phiKZ tail sheath contraction was found to be similar to that of phage T4.► The crystal structure of the phiKZ tail sheath protein fragment was determined ► Structural conservation was demonstrated among four tail sheath proteins ► There is little sequence similarity among these tail sheath proteins ► The structural rearrangement during contraction among these viruses is similar

Structure of a Blinkin-BUBR1 Complex Reveals an Interaction Crucial for Kinetochore-Mitotic Checkpoint Regulation via an Unanticipated Binding Site by Victor M. Bolanos-Garcia; Tiziana Lischetti; Dijana Matak-Vinković; Ernesto Cota; Pete J. Simpson; Dimitri Y. Chirgadze; David R. Spring; Carol V. Robinson; Jakob Nilsson; Tom L. Blundell (1895).

Crystal Structures of the Armadillo Repeat Domain of Adenomatous Polyposis Coli and Its Complex with the Tyrosine-Rich Domain of Sam68 by Ella Czarina Morishita; Kazutaka Murayama; Miyuki Kato-Murayama; Yoshiko Ishizuka-Katsura; Yuri Tomabechi; Tomoatsu Hayashi; Takaho Terada; Noriko Handa; Mikako Shirouzu; Tetsu Akiyama; Shigeyuki Yokoyama (1896).