Structure (v.19, #10)

In This Issue (v-vi).

Structure Meets the Membrane by Chris Lima; Andrej Sali; Milka Kostic (1347).

Show Me the MUN-y by Mary Munson (1348-1349).
The structure of the MUN domain of the synaptic protein Munc13-1 by Li et al., in this issue of Structure, shows that seemingly disparate regulators of SNARE-mediated membrane fusion are highly conserved at the structural level.

PDZ-Peptide Complexes: As Exciting as Ever by Gilles Trave (1350-1351).
In this issue, Babault et al. present a structural analysis of the PDZ domain of the PTPN4 phosphatase, free and bound to target peptides. This allows them to enhance the cell-killing properties of a pro-apoptotic peptide dramatically.

PARG: A Macrodomain in Disguise by Markus Hassler; Gytis Jankevicius; Andreas G. Ladurner (1351-1353).
Our understanding of poly-ADP-ribosylation as a posttranslational modification was limited by the lack of structural information on poly-ADP-ribose (PAR) hydrolysing enzymes. A recent study in Nature () reports the structure of PAR glycohydrolase (PARG), revealing unexpected similarity to the ubiquitous ADP-ribose-binding macrodomains.

It Takes Two to Get3 by Irmgard Sinning; Gert Bange; Klemens Wild (1353-1355).
Tail-anchored (TA) membrane proteins perform essential cellular functions. They are posttranslationally inserted into the endoplasmic reticulum (ER) membrane by interaction of the Get3 chaperone with the Get1/2 receptor. Two independent structural and functional analyses of the Get3/receptor complex by Stefer et al. and Mariappan et al. now provide insights into TA protein insertion.

Mechanosensitive Channels: What Can They Do and How Do They Do It? by Elizabeth S. Haswell; Rob Phillips; Douglas C. Rees (1356-1369).
While mechanobiological processes employ diverse mechanisms, at their heart are force-induced perturbations in the structure and dynamics of molecules capable of triggering subsequent events. Among the best characterized force-sensing systems are bacterial mechanosensitive channels. These channels reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane; the membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal, interpreted for the cell by mechanosensitive channels. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension or other functions. This review focuses on the bacterial mechanosensitive channels of large (MscL) and small (MscS) conductance and their eukaryotic homologs, with an emphasis on the outstanding issues surrounding the function and mechanism of this fascinating class of molecules.

Excitatory synaptic transmission in the brain is mediated by ligand-gated ion channels (iGluRs) activated by glutamate. Distinct from other neurotransmitter receptors, the extracellular domains of iGluRs are loosely packed assemblies with two clearly distinct layers, each of which has both local and global 2-fold axes of symmetry. By contrast, the iGluR transmembrane segments have 4-fold symmetry and share a conserved pore loop architecture found in tetrameric voltage-gated ion channels. The striking layered architecture of iGluRs revealed by the 3.6 Å resolution structure of an AMPA receptor homotetramer likely arose from gene fusion events that occurred early in evolution. Although this modular design has greatly facilitated biophysical and structural studies on individual iGluR domains, and suggested conserved mechanisms for iGluR gating, recent work is beginning to reveal unanticipated diversity in the structure, allosteric regulation, and assembly of iGluR subtypes.

Advances in Structural and Functional Analysis of Membrane Proteins by Electron Crystallography by Goragot Wisedchaisri; Steve L. Reichow; Tamir Gonen (1381-1393).
Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.

A New Generation of Crystallographic Validation Tools for the Protein Data Bank by Randy J. Read; Paul D. Adams; W. Bryan Arendall; Axel T. Brunger; Paul Emsley; Robbie P. Joosten; Gerard J. Kleywegt; Eugene B. Krissinel; Thomas Lütteke; Zbyszek Otwinowski; Anastassis Perrakis; Jane S. Richardson; William H. Sheffler; Janet L. Smith; Ian J. Tickle; Gert Vriend; Peter H. Zwart (1395-1412).
This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.► Validation criteria used by the PDB for X-ray crystal structures have been reassessed ► Key scores should be presented prominently in an easily understood format ► A concise validation report should be available to referees of papers on crystal structures

The Structure of a Tetrahydrofolate-Sensing Riboswitch Reveals Two Ligand Binding Sites in a Single Aptamer by Jeremiah J. Trausch; Pablo Ceres; Francis E. Reyes; Robert T. Batey (1413-1423).
Transport and biosynthesis of folate and its derivatives are frequently controlled by the tetrahydrofolate (THF) riboswitch in Firmicutes. We have solved the crystal structure of the THF riboswitch aptamer in complex with folinic acid, a THF analog. Uniquely, this structure reveals two molecules of folinic acid binding to a single structured domain. These two sites interact with ligand in a similar fashion, primarily through recognition of the reduced pterin moiety. 7-deazaguanine, a soluble analog of guanine, binds the riboswitch with nearly the same affinity as its natural effector. However, 7-deazaguanine effects transcriptional termination to a substantially lesser degree than folinic acid, suggesting that the cellular guanine pool does not act upon the THF riboswitch. Under physiological conditions the ligands display strong cooperative binding, with one of the two sites playing a greater role in eliciting the regulatory response, which suggests that the second site may play another functional role.► Crystal structure of the tetrahydrofolate (THF) riboswitch reveals two ligand sites ► Binding of the two ligands is highly cooperative at physiological magnesium ► Guanine binds to the THF riboswitch but does not effect regulation ► One THF binding site is more effective in regulating gene expression

Ligand-Dependent Perturbation of the Conformational Ensemble for the GPCR β2 Adrenergic Receptor Revealed by HDX by Graham M. West; Ellen Y.T. Chien; Vsevolod Katritch; Jovylyn Gatchalian; Michael J. Chalmers; Raymond C. Stevens; Patrick R. Griffin (1424-1432).
Mechanism of G protein-coupled receptor (GPCR) activation and their modulation by functionally distinct ligands remains elusive. Using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry, we examined the ligand-induced changes in conformational states and stability within the beta-2-adrenergic receptor (β2AR). Differential HDX reveals ligand-specific alterations in the energy landscape of the receptor's conformational ensemble. The inverse agonists timolol and carazolol were found to be most stabilizing even compared with the antagonist alprenolol, notably in intracellular regions where G proteins are proposed to bind, while the agonist isoproterenol induced the largest degree of conformational mobility. The partial agonist clenbuterol displayed conformational effects found in both the inverse agonists and the agonist. This study highlights the regional plasticity of the receptor and characterizes unique conformations spanning the entire receptor sequence stabilized by functionally selective ligands, all of which differ from the profile for the apo receptor.Display Omitted► Comprehensive HDX study comparing apo β2AR to β2AR in complex with five ligands ► Each complex affords a unique HDX fingerprint ► Inverse agonists and an antagonist show varying degrees of increased stability ► Full agonists increase conformational mobility in helix VIII on long timescales

Wnt Antagonists Bind through a Short Peptide to the First β-Propeller Domain of LRP5/6 by Eric Bourhis; Weiru Wang; Christine Tam; Jiyoung Hwang; Yingnan Zhang; Didier Spittler; Oscar W. Huang; Yan Gong; Alberto Estevez; Inna Zilberleyb; Lionel Rouge; Cecilia Chiu; Yan Wu; Mike Costa; Rami N. Hannoush; Yvonne Franke; Andrea G. Cochran (1433-1442).
The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations.► First structure of any domain from Wnt coreceptors LRP5/6 (LRP6 E1 β-propeller) ► The E1 propeller binds a short peptide present in Wnt inhibitors SOST and DKK1 ► The LRP5/6 binding motif is required for DKK1 and SOST function ► Human bone mineral density mutations in LRP5 disrupt critical peptide contacts

The Crystal Structure of a Munc13 C-terminal Module Exhibits a Remarkable Similarity to Vesicle Tethering Factors by Wei Li; Cong Ma; Rong Guan; Yibin Xu; Diana R. Tomchick; Josep Rizo (1443-1455).
Unc13/Munc13s play a crucial function in neurotransmitter release through their MUN domain, which mediates the transition from the Syntaxin-1/Munc18-1 complex to the SNARE complex. The MUN domain was suggested to be related to tethering factors, but no MUN-domain structure is available to experimentally validate this notion and address key unresolved questions about the interactions and minimal structural unit required for Unc13/Munc13 function. Here we identify an autonomously folded module within the MUN domain (MUN-CD) and show that its crystal structure is remarkably similar to several tethering factors. We also show that the activity in promoting the Syntaxin-1/Munc18-1 to SNARE complex transition is strongly impaired in MUN-CD. These results show that MUN domains and tethering factors indeed belong to the same family and may have a common role in membrane trafficking. We propose a model whereby the MUN-CD module is central for Munc13 function but full activity requires adjacent sequences.► The crystal structure of a C-terminal module of Munc13-1 (MUN-CD) is described ► MUN-CD is at the heart of Munc13 function, but full activity requires adjacent modules ► The structure of MUN-CD is very similar to those of diverse tethering factors ► MUN-domain containing proteins and tethering factors form a protein family

Crystal Structure of the HCV IRES Central Domain Reveals Strategy for Start-Codon Positioning by Katherine E. Berry; Shruti Waghray; Stefanie A. Mortimer; Yun Bai; Jennifer A. Doudna (1456-1466).
Translation of hepatitis C viral proteins requires an internal ribosome entry site (IRES) located in the 5′ untranslated region of the viral mRNA. The core domain of the hepatitis C virus (HCV) IRES contains a four-way helical junction that is integrated within a predicted pseudoknot. This domain is required for positioning the mRNA start codon correctly on the 40S ribosomal subunit during translation initiation. Here, we present the crystal structure of this RNA, revealing a complex double-pseudoknot fold that establishes the alignment of two helical elements on either side of the four-helix junction. The conformation of this core domain constrains the open reading frame's orientation for positioning on the 40S ribosomal subunit. This structure, representing the last major domain of HCV-like IRESs to be determined at near-atomic resolution, provides the basis for a comprehensive cryoelectron microscopy-guided model of the intact HCV IRES and its interaction with 40S ribosomal subunits.Display Omitted► Crystal structure of the most complex HCV IRES domain reveals its functional core ► A tertiary base pair creates a double pseudoknot encompassing a four-helix junction ► Final domain to be solved facilitates comprehensive IRES model bound to 40S subunit ► Orientation of two helical elements directs start-codon positioning by the IRES

Crystal Structure of a BCL-W Domain-Swapped Dimer: Implications for the Function of BCL-2 Family Proteins by Erinna F. Lee; Grant Dewson; Brian J. Smith; Marco Evangelista; Anne Pettikiriarachchi; Con Dogovski; Matthew A. Perugini; Peter M. Colman; W. Douglas Fairlie (1467-1476).
The prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.► Crystal structure of a dimer of BCL-W described ► A major conformational change not seen in BCL-2 protein structures is revealed ► A functional role for the α4-α5 hinge is demonstrated

A Helix Heterodimer in a Lipid Bilayer: Prediction of the Structure of an Integrin Transmembrane Domain via Multiscale Simulations by Antreas C. Kalli; Benjamin A. Hall; Iain D. Campbell; Mark S.P. Sansom (1477-1484).
Display Omitted► Multiscale simulation have been used to model transmembrane helix heterodimers ► This method is applied to the integrin αIIb/β3 heterodimer ► The model exhibits right-handed packing of the helices, in agreement with NMR structures ► Flexibility of the αIIb/β3 interface suggests of a role for alternative packing modes in transbilayer signaling.

The Phe105 Loop of Alix Bro1 Domain Plays a Key Role in HIV-1 Release by Paola Sette; Ruiling Mu; Vincent Dussupt; Jiansheng Jiang; Greg Snyder; Patrick Smith; Tsan Sam Xiao; Fadila Bouamr (1485-1495).
Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical “boomerang” folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.► High-resolution structures of Alix Bro1 domain paralogs HD-PTP and Brox ► Phe105 loop is a unique structural feature in the Bro1 domain of Alix ► Mutation of residues within the Phe105 loop compromise Alix function in HIV-1 release ► Phe105 loop is a new functional determinant of Alix

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 (1496-1508).
Adenomatous polyposis coli (APC) is a tumor suppressor protein commonly mutated in colorectal tumors. APC plays important roles in Wnt signaling and other cellular processes. Here, we present the crystal structure of the armadillo repeat (Arm) domain of APC, which facilitates the binding of APC to various proteins. APC-Arm forms a superhelix with a positively charged groove. We also determined the structure of the complex of APC-Arm with the tyrosine-rich (YY) domain of the Src-associated in mitosis, 68 kDa protein (Sam68), which regulates TCF-1 alternative splicing. Sam68-YY forms numerous interactions with the residues on the groove and is thereby fixed in a bent conformation. We assessed the effects of mutations and phosphorylation on complex formation between APC-Arm and Sam68-YY. Structural comparisons revealed different modes of ligand recognition between the Arm domains of APC and other Arm-containing proteins.Display Omitted► Crystal structures of the armadillo domain of APC and its complex with Sam68 ► The armadillo domain of APC forms a superhelix with a positively charged groove ► The tyrosine-rich domain of Sam68 is bound in a bent conformation to the APC groove ► Mutagenesis defines the residues that are critical for APC⋅Sam68 complex formation

Structural Basis of the Ca2+ Inhibitory Mechanism of Drosophila Na+/Ca2+ Exchanger CALX and Its Modification by Alternative Splicing by Mousheng Wu; Shuilong Tong; Jennifer Gonzalez; Vasanthi Jayaraman; John L. Spudich; Lei Zheng (1509-1517).
The Na+/Ca2+ exchanger CALX promotes Ca2+ efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca2+ homeostasis. CALX activity is negatively regulated by specific Ca2+ interaction within its two intracellular Ca2+ regulatory domains CBD1 and CBD2, yet how the Ca2+ binding is converted to molecular motion to operate the exchanger is unknown. Here, we report crystal structures of the entire Ca2+ regulatory domain CBD12 from two alternative splicing isoforms, CALX 1.1 and 1.2, exhibiting distinct regulatory Ca2+ dependency. The structures show an open V-shaped conformation with four Ca2+ ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca2+-binding analysis support that the Ca2+ inhibition of CALX is achieved by interdomain conformational changes induced by Ca2+ binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca2+ regulatory property of the exchangers.Display Omitted► Crystal structures of the complete Ca2+ regulatory domain of the exchanger CALX ► Ca2+ binding at the domain interface between CBD1 and CBD2 ► Alternative splicing induces CBD domain orientation angle change ► Implication of the Ca2+ regulatory mechanism of the Na+/Ca2+ exchanger family

Peptides Targeting the PDZ Domain of PTPN4 Are Efficient Inducers of Glioblastoma Cell Death by Nicolas Babault; Florence Cordier; Mireille Lafage; Joseph Cockburn; Ahmed Haouz; Christophe Prehaud; Félix A. Rey; Muriel Delepierre; Henri Buc; Monique Lafon; Nicolas Wolff (1518-1524).
PTPN4, a human tyrosine phosphatase, protects cells against apoptosis. This protection could be abrogated by targeting the PDZ domain of this phosphatase with a peptide mimicking the C-terminal sequence of the G protein of an attenuated rabies virus strain. Here, we demonstrate that glioblastoma death is triggered upon intracellular delivery of peptides, either from viral origin or from known endogenous ligands of PTPN4-PDZ, such as the C terminus sequence of the glutamate receptor subunit GluN2A. The killing efficiency of peptides closely reflects their affinities for the PTPN4-PDZ. The crystal structures of two PTPN4-PDZ/peptide complexes allow us to pinpoint the main structural determinants of binding and to synthesize a peptide of high affinity for PTPN4-PDZ enhancing markedly its cell death capacity. These results allow us to propose a potential mechanism for the efficiency of peptides and provide a target and a robust framework for the design of new pro-death compounds.► The structures of PTPN4-PDZ complexed to viral and endogenous peptides are reported ► Slight changes in the PDZ/viral ligands contacts lead to drastic functional effect ► The killing efficiency of peptides closely reflects their affinities for the PDZ ► The best optimized sequence for PTPN4-PDZ binding is the best inducer of cell death

Structural Insight into the Mycobacterium tuberculosis Rv0020c Protein and Its Interaction with the PknB Kinase by Christian Roumestand; Jade Leiba; Nathalie Galophe; Emmanuel Margeat; André Padilla; Yannick Bessin; Philippe Barthe; Virginie Molle; Martin Cohen-Gonsaud (1525-1534).
The protein Rv0020c from Mycobacterium tuberculosis, also called FhaA, is one of the major substrates of the essential Ser/Thr protein kinase (STPK) PknB. The protein is composed of three domains and is phosphorylated on a unique site in its N terminus. We solved the solution structure of both N- and C-terminal domains and demonstrated that the approximately 300 amino acids of the intermediate domain are not folded. We present evidence that the FHA, a phosphospecific binding domain, of Rv0020c does not interact with the phosphorylated catalytic domains of PknB, but with the phosphorylated juxtamembrane domain that links the catalytic domain to the mycobacterial membrane. We also demonstrated that the degree and the pattern of phosphorylation of this juxtamembrane domain modulates the affinity of the substrate (Rv0020c) toward its kinase (PknB).► Full structural characterization of the three-domain protein Rv0020c ► Rv0020c interacts with the juxtamembrane domain of PknB and not with the catalytic domain ► Rv0020c/PknB interaction is tuned by phosphorylation pattern of PknB