Structure (v.16, #10)

In This Issue (ix-x).

Key Determinants of Rab Specificity by Aymelt Itzen; Roger S. Goody (1437-1439).
Structures are reported for complexes between Rab27 and the Rab27-binding domains of two molecules involved in successive steps in melanosome transport. Comparison of the Rab27B:Slac2-a () and Rab27A:Slp2-a () structures with that of the Rab3A:Rabphilin complex gives important clues to the specificity of the interactions.

Tripping a Switch: PDZRhoGEF rgRGS-Bound Gα13 by Mehdi Bagheri Hamaneh; Matthias Buck (1439-1441).
Ras superfamily guanine nucleotide-binding proteins, such as G-proteins and small GTPases, are a paradigm for two-state molecular switches in cell signaling. Recent experimental and theoretical studies question this simple model. Now provide evidence that the rgRGS domain of PDZRhoGEF has evolved to “trip” the nucleotide-dependent switch of the subunit of the heterotrimeric G-protein, Gα13, so that it can also bind to its GDP state.

A Pilot Sheds Light on Secretin Assembly by Jeremy Derrick (1441-1442).
The structure of a pilot protein (MxiM), in complex with a peptide from its cognate secretin (MxiD), reported by , shows how specific recognition between the two proteins is achieved and suggests a model for the way in which pilot proteins may function.

Chaperone-Assisted Crystallography with DARPins by Gaby Sennhauser; Markus G. Grütter (1443-1453).
The structure of proteins that are difficult to crystallize can often be solved by forming a noncovalent complex with a helper protein—a crystallization “chaperone.” Although several such applications have been described to date, their handling usually is still very laborious. A valuable addition to the present repertoire of binding proteins is the recently developed designed ankyrin repeat protein (DARPin) technology. DARPins are built based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein. The broad potential of these binding proteins for X-ray crystallography is illustrated by five cocrystal structures that have been determined recently comprising target proteins from distinct families, namely a sugar binding protein, two kinases, a caspase, and a membrane protein. This article reviews the opportunities of this technology for structural biology and the structural aspects of the DARPin-protein complexes.
Keywords: PROTEINS;

Structures of CaV2 Ca2+/CaM-IQ Domain Complexes Reveal Binding Modes that Underlie Calcium-Dependent Inactivation and Facilitation by Eun Young Kim; Christine H. Rumpf; Yuichiro Fujiwara; Elizabeth S. Cooley; Filip Van Petegem; Daniel L. Minor (1455-1467).
Calcium influx drives two opposing voltage-activated calcium channel (CaV) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca2+/calmodulin (Ca2+/CaM) lobes produce CDI and CDF through interactions with the CaVα1 subunit IQ domain. Curiously, Ca2+/CaM lobe modulation polarity appears inverted between CaV1s and CaV2s. Here, we present crystal structures of CaV2.1, CaV2.2, and CaV2.3 Ca2+/CaM-IQ domain complexes. All display binding orientations opposite to CaV1.2 with a physical reversal of the CaM lobe positions relative to the IQ α-helix. Titration calorimetry reveals lobe competition for a high-affinity site common to CaV1 and CaV2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal CaV2 Ca2+/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of CaV feedback modulation and indicate that CaV1 and CaV2 IQ domains bear a dedicated CDF site that exchanges Ca2+/CaM lobe occupants.

Elucidation of Rab27 Recruitment by Its Effectors: Structure of Rab27a Bound to Exophilin4/Slp2-a by Leonard M.G. Chavas; Kentaro Ihara; Masato Kawasaki; Seiji Torii; Tamami Uejima; Ryuichi Kato; Tetsuro Izumi; Soichi Wakatsuki (1468-1477).
Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. Rab27a regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 Å resolution structure of Rab27a in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of Rab27a, and specific affinity toward Rab27a is modulated by a shift in the orientation of the effector structural motif (S/T)(G/L)xW(F/Y)2. The observed structural complementation between the interacting surfaces of Rab27a and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.
Keywords: SIGNALING;

Structural Basis for the Exclusive Specificity of Slac2-a/Melanophilin for the Rab27 GTPases by Mutsuko Kukimoto-Niino; Ayako Sakamoto; Eiko Kanno; Kyoko Hanawa-Suetsugu; Takaho Terada; Mikako Shirouzu; Mitsunori Fukuda; Shigeyuki Yokoyama (1478-1490).
Rab27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/melanophilin. Mutations that disrupt the Rab27A/Slac2-a interaction cause human Griscelli syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of Rab27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A Rab27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.
Keywords: SIGNALING;

Virus Capsid Expansion Driven by the Capture of Mobile Surface Loops by Kelly K. Lee; Lu Gan; Hiro Tsuruta; Crystal Moyer; James F. Conway; Robert L. Duda; Roger W. Hendrix; Alasdair C. Steven; John E. Johnson (1491-1502).
The capsids of tailed-DNA bacteriophages first assemble as procapsids, which mature by converting into a new form that is strong enough to contain a densely packed viral chromosome. We demonstrate that the intersubunit crosslinking that occurs during maturation of HK97 capsids actually promotes the structural transformation. Small-angle X-ray scattering and crosslinking assays reveal that a shift in the crosslink pattern accompanies conversion of a semimature particle, Expansion Intermediate-I/II, to a more mature state, Balloon. This transition occurs in a switch-like fashion. We find that crosslink formation shifts the global conformational balance to favor the balloon state. A pseudoatomic model of EI-I/II derived from cryo-EM provides insight into the relationship between crosslink formation and conformational switching.

Histone acetylation plays important roles for the regulation of many fundamental cellular processes. Saccharomyces cerevisiae Rtt109 is an important class of histone acetyltransferases (HATs), which promote genome stability by directly acetylating newly synthesized histone H3 lysine 56 (H3-K56) through an unknown mechanism. Here, we report the crystal structures of Rtt109 at 2.2 Å and Rtt109/Acetyl-CoA binary complex at 1.9 Å. The structure displays a vise-like topology with mixed three-layered α/β module forming the central module, whose core region resembles the structure of GCN5 HAT domain and P300/CBP HAT domain. Using structural and biochemical analyses, we have discovered the catalytic active site and have identified Asp288 as the deprotonation residue and Lys290 as the autoacetylation residue. We have further proposed the unique H3-K56 anchoring pocket and the potential H3αN binding groove. Our work has provided structural insights to understand the acetylation mechanism of H3-K56 by Rtt109.
Keywords: DNA; PROTEINS;

Architecture of the Pontin/Reptin Complex, Essential in the Assembly of Several Macromolecular Complexes by Eva Torreira; Sudhakar Jha; José R. López-Blanco; Ernesto Arias-Palomo; Pablo Chacón; Cristina Cañas; Sylvia Ayora; Anindya Dutta; Oscar Llorca (1511-1520).
Pontin and reptin belong to the AAA+ family, and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 Å. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared with the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different than previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins.

Structural Analysis of the Interactions Between Paxillin LD Motifs and α-Parvin by Sonja Lorenz; Ioannis Vakonakis; Edward D. Lowe; Iain D. Campbell; Martin E.M. Noble; Maria K. Hoellerer (1521-1531).
The adaptor protein paxillin contains five conserved leucine-rich (LD) motifs that interact with a variety of focal adhesion proteins, such as α-parvin. Here, we report the first crystal structure of the C-terminal calponin homology domain (CHC) of α-parvin at 1.05 Å resolution and show that it is able to bind all the LD motifs, with some selectivity for LD1, LD2, and LD4. Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on α-parvin-CHC, which is located at the rim of the canonical fold and includes part of the inter-CH domain linker. Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations. Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/α-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.
Keywords: PROTEINS;

Recognition of the Activated States of Gα13 by the rgRGS Domain of PDZRhoGEF by Zhe Chen; William D. Singer; Shahab M. Danesh; Paul C. Sternweis; Stephen R. Sprang (1532-1543).
G12 class heterotrimeric G proteins stimulate RhoA activation by RGS-RhoGEFs. However, p115RhoGEF is a GTPase Activating Protein (GAP) toward Gα13, whereas PDZRhoGEF is not. We have characterized the interaction between the PDZRhoGEF rgRGS domain (PRG-rgRGS) and the alpha subunit of G13 and have determined crystal structures of their complexes in both the inactive state bound to GDP and the active states bound to GDP•AlF (transition state) and GTPγS (Michaelis complex). PRG-rgRGS interacts extensively with the helical domain and the effector-binding sites on Gα13 through contacts that are largely conserved in all three nucleotide-bound states, although PRG-rgRGS has highest affinity to the Michaelis complex. An acidic motif in the N terminus of PRG-rgRGS occupies the GAP binding site of Gα13 and is flexible in the GDP•AlF complex but well ordered in the GTPγS complex. Replacement of key residues in this motif with their counterparts in p115RhoGEF confers GAP activity.

Structural Characterization of the Type-III Pilot-Secretin Complex from Shigella flexneri by Mark Okon; Trevor F. Moraes; Paula I. Lario; A. Louise Creagh; Charles A. Haynes; Natalie C.J. Strynadka; Lawrence P. McIntosh (1544-1554).
Assembly of the type-III secretion apparatus, which translocates proteins through both membranes of Gram-negative bacterial pathogens into host cells, requires the formation of an integral outer-membrane secretin ring. Typically, a small lipidated pilot protein is necessary for the stabilization and localization of this ring. Using NMR spectroscopy, we demonstrate that the C-terminal residues 553–570 of the Shigella flexneri secretin MxiD encompass the minimal binding domain for its cognate pilot MxiM. Although unstructured in isolation, upon complex formation with MxiM, these residues fold into an amphipathic turn-helix motif that caps the elongated hydrophobic cavity of the cracked β-barrel pilot. Along with a rearrangement of core aromatic residues, this prevents the binding of lipids within the cavity. The mutually exclusive association of lipids and MxiD with MxiM establishes a framework for understanding the role of a pilot in the outer-membrane insertion and multimerization of the secretin ring.

Structure of Seneca Valley Virus-001: An Oncolytic Picornavirus Representing a New Genus by Sangita Venkataraman; Seshidhar P. Reddy; Jackie Loo; Neeraja Idamakanti; Paul L. Hallenbeck; Vijay S. Reddy (1555-1561).
The crystal structure of Seneca Valley Virus-001 (SVV-001), the representative member of a new genus, Senecavirus, is reported at 2.3Å resolution. SVV-001 is the first naturally occurring nonpathogenic picornavirus shown to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features. The nonsegmented (+) ssRNA genome of SVV-001 shares closest sequence similarity with the genomes of the members of Cardiovirus. The overall tertiary structure of VP1-VP4 subunits is conserved with the exception of loops, especially those of VP1 that show large deviations relative to the members of the cardioviruses. The surface loops of VP1 and VP2 are predicted to mediate cell tropism of SVV-001. In addition, the organization of the packaged nucleic acid density indicates that certain regions of VP2 and VP4 interact closely with the packaged nucleic acid.

Structural Basis for Ca2+-Dependent Formation of ALG-2/Alix Peptide Complex: Ca2+/EF3-Driven Arginine Switch Mechanism by Hironori Suzuki; Masato Kawasaki; Tatsutoshi Inuzuka; Mayumi Okumura; Takeshi Kakiuchi; Hideki Shibata; Soichi Wakatsuki; Masatoshi Maki (1562-1573).
ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca2+-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn2+-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn2+-bound form. Binding of Ca2+ to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca2+/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.

Structure and Plasticity of Endophilin and Sorting Nexin 9 by Qi Wang; Hung Yi Kristal Kaan; Reshma Noordin Hooda; Shih Lin Goh; Holger Sondermann (1574-1587).
Endophilin and Sorting Nexin 9 (Snx9) play key roles in endocytosis by membrane curvature sensing and remodeling via their Bin/Amphiphysin/Rvs (BAR) domains. BAR and the related F-BAR domains form dimeric, crescent-shaped units that occur N- or C-terminally to other lipid-binding, adaptor, or catalytic modules. In crystal structures, the PX-BAR unit of Snx9 (Snx9PX-BAR) adopts an overall compact, moderately curved conformation. SAXS-based solution studies revealed an alternative, more curved state of Snx9PX-BAR in which the PX domains are flexibly connected to the BAR domains, providing a model for how Snx9 exhibits lipid-dependent curvature preferences. In contrast, Endophilin appears to be rigid in solution, and the SH3 domains are located at the distal tips of a BAR domain dimer with fixed curvature. We also observed tip-to-tip interactions between the BAR domains in a trigonal crystal form of Snx9PX-BAR reminiscent of functionally important interactions described for F-BAR domains.

A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate by Jorge E. Zuniga; James J. Schmidt; Timothy Fenn; James C. Burnett; Demet Araç; Rick Gussio; Robert G. Stafford; Shirin S. Badie; Sina Bavari; Axel T. Brunger (1588-1597).
Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (Ki = 41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the “proton shuttle” E224. This mechanism of inhibition is aided by residue contacts in the conserved S1′ pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2′ residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.