Crystal structure of Rnd3/RhoE: functional implications

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up-regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that ms1 may be important in striated muscle biology and the development of pressure-induced left ventricular hypertrophy.     

Crystal structure of an activated variant of small heat shock protein Hsp16.5

How does the sequence of a single small heat shock protein (sHSP) assemble into oligomers of different sizes? To gain insight into the underlying structural mechanism, we determined the crystal structure of an engineered variant of Methanocaldococcus jannaschii Hsp16.5 wherein a 14 amino acid peptide from human heat shock protein 27 (Hsp27) was inserted at the junction of the N-terminal region and the α-crystallin domain. In response to this insertion, the oligomer shell expands from 24 to 48 subunits while maintaining octahedral symmetry. Oligomer rearrangement does not alter the fold of the conserved α-crystallin domain nor does it disturb the interface holding the dimeric building block together. Rather, the flexible C-terminal tail of Hsp16.5 changes its orientation relative to the α-crystallin domain which enables alternative packing of dimers. This change in orientation preserves a peptide-in-groove interaction of the C-terminal tail with an adjacent β-sandwich, thereby holding the assembly together. The interior of the expanded oligomer, where substrates presumably bind, retains its predominantly nonpolar character relative to the outside surface. New large windows in the outer shell provide increased access to these substrate-binding regions, thus accounting for the higher affinity of this variant to substrates. Oligomer polydispersity regulates sHSPs chaperone activity in vitro and has been implicated in their physiological roles. The structural mechanism of Hsp16.5 oligomer flexibility revealed here, which is likely to be highly conserved across the sHSP superfamily, explains the relationship between oligomer expansion observed in disease-linked mutants and changes in chaperone activity.     

Crystal structure of the catalytic domain of a human thioredoxin-like protein.

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N-terminal catalytic domain (hTRXL-N) of hTRXL in its oxidized form at 2.2-A resolution. Although this domain shares a similar three-dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL-N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL-N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL-N suggest that it could interact with the acidic amino-acid rich C-terminal region, thereby suggesting a novel regulation mechanism.     

Crystal structure of the ligand binding domain of netrin G2

Netrin G proteins represent a small family of synaptic cell adhesion molecules related to netrins and to the polymerization domains of laminins. Two netrin G proteins are encoded in vertebrate genomes, netrins G1 and G2, which are known to bind the leucine-rich repeat proteins netrin G ligand (NGL)-1 and NGL-2, respectively. Netrin G proteins share a common multi-domain architecture comprising a laminin N-terminal (LN) domain followed by three laminin epidermal growth factor-like (LE) domains and a C′ region containing a glycosylphosphatidylinositol anchor. Here, we use deletion analysis to show that the LN domain region of netrin Gs contains the binding site for NGLs to which they bind with 1:1 stoichiometry and sub-micromolar affinity. Netrin Gs are alternatively spliced in their LE domain regions, but the binding region, the LN domain, is identical in all splice forms. We determined the crystal structure for a fragment comprising the LN domain and domain LE1 of netrin G2 by sulfur single-wavelength anomalous diffraction phasing and refined it to 1.8 Å resolution. The structure reveals an overall architecture similar to that of laminin α chain LN domains but includes significant differences including a Ca²⁺ binding site in the LN domain. These results reveal the minimal binding unit for interaction of netrin Gs with NGLs, define structural features specific to netrin Gs, and suggest that netrin G alternative splicing is not involved in NGL recognition.     

Crystal structure of an Fab fragment in complex with a meningococcal serosubtype antigen and a protein G domain.

Many pathogens present highly variable surface proteins to their host as a means of evading immune responses. The structure of a peptide antigen corresponding to the subtype P1.7 variant of the porin PorA from the human pathogen Neisseria meningitidis was determined by solution of the X-ray crystal structure of the ternary complex of the peptide (ANGGASGQVK) in complex with a Fab fragment and a domain from streptococcal protein G to 1.95 A resolution. The peptide adopted a beta-hairpin structure with a type I beta-turn between residues Gly4P and Gly7P, the conformation of the peptide being further stabilised by a pair of hydrogen bonds from the side-chain of Asn2P to main-chain atoms in Val9P. The antigen binding site within the Fab formed a distinct crevice lined by a high proportion of apolar amino acids. Recognition was supplemented by hydrogen bonds from heavy chain residues Thr50H, Asp95H, Leu97H and Tyr100H to main-chain and side-chain atoms in the peptide. Complementarity-determining region (CDR) 3 of the heavy chain was responsible for approximately 50 % of the buried surface area formed by peptide-Fab binding, with the remainder made up from CDRs 1 and 3 of the light chain and CDRs 1 and 2 of the heavy chain. Knowledge of the structures of variable surface antigens such as PorA is an essential prerequisite to a molecular understanding of antigenic variation and its implications for vaccine design.     

Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.     

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen closed conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.     

Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A

The small G protein Rab3A plays an important role in the regulation of neurotransmitter release. The crystal structure of activated Rab3A/GTP/Mg2+ bound to the effector domain of rabphilin-3A was solved to 2.6 A resolution. Rabphilin-3A contacts Rab3A in two distinct areas. The first interface involves the Rab3A switch I and switch II regions, which are sensitive to the nucleotide-binding state of Rab3A. The second interface consists of a deep pocket in Rab3A that interacts with a SGAWFF structural element of rabphilin-3A. Sequence and structure analysis, and biochemical data suggest that this pocket, or Rab complementarity-determining region (RabCDR), establishes a specific interaction between each Rab protein and its effectors. RabCDRs could be major determinants of effector specificity during vesicle trafficking and fusion.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

Crystal structure of the actin binding domain of the cyclase-associated protein

Cyclase-associated protein (CAP or Srv2p) is a modular actin monomer binding protein that directly regulates filament dynamics and has been implicated in a number of complex developmental and morphological processes, including mRNA localization and the establishment of cell polarity. The crystal structure of the C-terminal dimerization and actin monomer binding domain (C-CAP) reveals a highly unusual dimer, composed of monomers possessing six coils of right-handed beta-helix flanked by antiparallel beta-strands. Domain swapping, involving the last two strands of each monomer, results in the formation of an extended dimer with an extensive interface. This structural and biochemical characterization provides new insights into the organization and potential mechanistic properties of the multiprotein assemblies that integrate dynamic actin processes into the overall physiology of the cell. An unanticipated finding is that the unique tertiary structure of the C-CAP monomer provides a structural model for a wide range of molecules, including RP2 and cofactor C, proteins involved in X-linked retinitis pigmentosa and tubulin maturation, respectively, as well as several uncharacterized proteins that exhibit very diverse domain organizations. Thus, the unusual right-handed beta-helical fold present in C-CAP appears to support a wide range of biological functions.     

Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site

During the maturation of rotaviral particles, non-structural protein 4 (NSP4) plays a critical role in the translocation of the immature capsid into the lumen of the endoplasmic reticulum. Full-length NSP4 and a 22 amino acid peptide (NSP4(114-135)) derived from this protein have been shown to induce diarrhea in young mice in an age-dependent manner, and may therefore be the agent responsible for rotavirally-induced symptoms. We have determined the crystal structure of the oligomerization domain of NSP4 which spans residues 95 to 137 (NSP4(95-137)). NSP4(95-137) self-associates into a parallel, tetrameric coiled-coil, with the hydrophobic core interrupted by three polar layers occupying a and d-heptad positions. Side-chains from two consecutive polar layers, consisting of four Gln123 and two of the four Glu120 residues, coordinate a divalent cation. Two independent structures built from MAD-phased data indicated the presence of a strontium and calcium ion bound at this site, respectively. This metal-binding site appears to play an important role in stabilizing the homo-tetramer, which has implications for the engagement of NSP4 as an enterotoxin.     

Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP

The protein kinase Akt/PKB is stimulated by the phosphorylation of two regulatory residues, Thr 309 of the activation segment and Ser 474 of the hydrophobic motif (HM), that are structurally and functionally conserved within the AGC kinase family. To understand the mechanism of PKB regulation, we determined the crystal structures of activated kinase domains of PKB in complex with a GSK3beta-peptide substrate and an ATP analog. The activated state of the kinase was generated by phosphorylating Thr 309 using PDK1 and mimicking Ser 474 phosphorylation either with the S474D substitution or by replacing the HM of PKB with that of PIFtide, a potent mimic of a phosphorylated HM. Comparison with the inactive PKB structure indicates that the role of Ser 474 phosphorylation is to promote the engagement of the HM with the N-lobe of the kinase domain, promoting a disorder-to-order transition of the alphaC helix. The alphaC helix, by interacting with pThr 309, restructures and orders the activation segment, generating an active kinase conformation. Analysis of the interactions between PKB and the GSK3beta-peptide explains how PKB selects for protein substrates distinct from those of PKA.     

Crystal structure of human ribosomal protein L10 core domain reveals eukaryote-specific motifs in addition to the conserved fold

A phylogenetically conserved ribosomal protein L16p/L10e organizes the architecture of the aminoacyl tRNA binding site on the large ribosomal subunit. Eukaryotic L10 also exhibits a variety of cellular activities, and, in particular, human L10 is known as a putative tumor suppressor, QM. We have determined the 2.5-Å crystal structure of the human L10 core domain that corresponds to residues 34-182 of the full-length 214 amino acids. Its two-layered α + β architecture is significantly similar to those of the archaeal and bacterial homologues, substantiating a high degree of structural conservation across the three phylogenetic domains. A cation-binding pocket formed between α2 and β6 is similar to that of the archaeal L10 protein but appears to be better ordered. Previously reported L10 mutations that cause defects in the yeast ribosome are clustered around this pocket, indicating that its integrity is crucial for its role in L10 function. Characteristic interactions among Arg90-Trp171-Arg139 guide the C-terminal part outside of the central fold, implying that the eukaryote-specific C-terminal extension localizes on the outer side of the ribosome.     

Crystal structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.     

Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Argonaute (AGO) proteins are core components of RNA‐induced silencing complexes and have essential roles in RNA‐mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino‐terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE‐2 from Neurospora crassa. This domain adopts a Rossmann‐like fold and recognizes the 5′‐terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5′‐nucleotide‐binding site shares common residues with a second, adjacent ligand‐binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.     

Crystal structure of the C-terminal domain of a flagellar hook-capping protein from Xanthomonas campestris

The crystal structure of the C-terminal domain of a hook-capping protein FlgD from the plant pathogen Xanthomonas campestris (Xc) has been determined to a resolution of ca 2.5 Å using X-ray crystallography. The monomer of whole FlgD comprises 221 amino acids with a molecular mass of 22.7 kDa, but the flexible N-terminus is cleaved for up to 75 residues during crystallization. The final structure of the C-terminal domain reveals a novel hybrid comprising a tudor-like domain interdigitated with a fibronectin type III domain. The C-terminal domain of XcFlgD forms three types of dimers in the crystal. In agreement with this, analytical ultracentrifugation and gel filtration experiments reveal that they form a stable dimer in solution. From these results, we propose that the Xc flagellar hook cap protein FlgD comprises two individual domains, a flexible N-terminal domain that cannot be detected in the current study and a stable C-terminal domain that forms a stable dimer.