Crystal structure of yeast V-ATPase subunit C reveals its stator function

Vacuolar H(+)-ATPase (V-ATPase) has a crucial role in the vacuolar system of eukaryotic cells. It provides most of the energy required for transport systems that utilize the proton-motive force that is generated by ATP hydrolysis. Some, but not all, of the V-ATPase subunits are homologous to those of F-ATPase and the nonhomologous subunits determine the unique features of V-ATPase. We determined the crystal structure of V-ATPase subunit C (Vma5p), which does not show any homology with F-ATPase subunits, at 1.75 A resolution. The structural features suggest that subunit C functions as a flexible stator that holds together the catalytic and membrane sectors of the enzyme. A second crystal form that was solved at 2.9 A resolution supports the flexible nature of subunit C. These structures provide a framework for exploring the unique mechanistic features of V-ATPases.     

Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes

The crystal structure of guanylate kinase (GK) from yeast (Saccharomyces cerevisiae) with a non-acetylated N terminus has been determined in its unligated form (apo-GK) as well as in complex with GMP (GK.GMP). The structure of apo-GK was solved with multiwavelength anomalous diffraction data and refined to an R-factor of 0.164 (R(free)=0.199) at 2.3 A resolution. The structure of GK.GMP was determined using the crystal structure of GK with an acetylated N terminus as the search model and refined to an R-factor of 0.156 (R(free)=0.245) at 1.9 A. GK belongs to the family of nucleoside monophosphate (NMP) kinases and catalyzes the reversible phosphoryl transfer from ATP to GMP. Like other NMP kinases, GK consists of three dynamic domains: the CORE, LID, and NMP-binding domains. Dramatic movements of the GMP-binding domain and smaller but significant movements of the LID domain have been revealed by comparing the structures of apo-GK and GK.GMP. apo-GK has a much more open conformation than the GK.GMP complex. Systematic analysis of the domain movements using the program DynDom shows that the large movements of the GMP-binding domain involve a rotation around an effective hinge axis approximately parallel with helix 3, which connects the GMP-binding and CORE domains. The C-terminal portion of helix 3, which connects to the CORE domain, has strikingly higher temperature factors in GK.GMP than in apo-GK, indicating that these residues become more mobile upon GMP binding. The results suggest that helix 3 plays an important role in domain movement. Unlike the GMP-binding domain, which moves toward the active center of the enzyme upon GMP binding, the LID domain moves away from the active center and makes the presumed ATP-binding site more open. Therefore, the LID domain movement may facilitate the binding of MgATP. The structure of the recombinant GK.GMP complex superimposes very well with that of the native GK.GMP complex, indicating that N-terminal acetylation does not have significant impact on the three-dimensional structure of GK.     

Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain

Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 angstroms crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel alpha-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the "leucine chain." The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.     

Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region

UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F⁶¹⁹ in the motif stacks partly over cis-Pro⁶²⁰ in one conformation, whereas it is swung out from cis-P⁶²⁰, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCP-binding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP.     

NMR structure of the heme chaperone CcmE reveals a novel functional motif

The concept of metal chaperones involves transient binding of metallic cofactors by specific proteins for delivery to enzymes in which they function. Metal chaperones thus provide a protective, as well as a transport, function. We report the first structure of a heme chaperone, CcmE, which comprises these two functions. We propose that the covalent attachment of heme to an exposed histidine occurs after heme binding at the surface of a rigid molecule with a flexible C-terminal domain. CcmE belongs to a family of proteins with a specific fold, which all share a function in delivery of specific molecular cargo.     

Crystal structure of a putative CN hydrolase from yeast

The crystal structure of a yeast hypothetical protein with sequence similarity to CN hydrolases has been determined to 2.4 A resolution by the multiwavelength anomalous dispersion (MAD) method. The protein folds as a four-layer alphabetabetaalpha sandwich and exists as a dimer in the crystal and in solution. It was selected in a structural genomics project as representative of CN hydrolases at a time when no structures had been determined for members of this family. Structures for two other members of the family have since been reported and the three proteins have similar topology and dimerization modes, which are distinct from those of other alphabetabetaalpha proteins whose structures are known. The dimers form an unusual eight-layer alphabetabetaalpha:alphabetabetaalpha structure. Although the precise enzymatic reactions catalyzed by the yeast protein are not known, considerable information about the active site may be deduced from conserved sequence motifs, comparative biochemical information, and comparison with known structures of hydrolase active sites. As with serine hydrolases, the active-site nucleophile (cysteine in this case) is positioned on a nucleophile elbow.     

Crystal structure of yeast Sco1

The Sco family of proteins are involved in the assembly of the dinuclear Cu_A site in cytochrome c oxidase (COX), the terminal enzyme in aerobic respiration. These proteins, which are found in both eukaryotes and prokaryotes, are characterized by a conserved CXXXC sequence motif that binds copper ions and that has also been proposed to perform a thiol:disulfide oxidoreductase function. The crystal structures of Saccharomyces cerevisiae apo Sco1 (apo-ySco1) and Sco1 in the presence of copper ions (Cu–ySco1) were determined to 1.8- and 2.3-Å resolutions, respectively. Yeast Sco1 exhibits a thioredoxin-like fold, similar to that observed for human Sco1 and a homolog from Bacillus subtilis. The Cu–ySco1 structure, obtained by soaking apo-ySco1 crystals in copper ions, reveals an unexpected copper-binding site involving Cys181 and Cys216, cysteine residues present in ySco1 but not in other homologs. The conserved CXXXC cysteines, Cys148 and Cys152, can undergo redox chemistry in the crystal. An essential histidine residue, His239, is located on a highly flexible loop, denoted the Sco loop, and can adopt positions proximal to both pairs of cysteines. Interactions between ySco1 and its partner proteins yeast Cox17 and yeast COX2 are likely to occur via complementary electrostatic surfaces. This high-resolution model of a eukaryotic Sco protein provides new insight into Sco copper binding and function.     

Crystal structure of the coxsackievirus A16 RNA-dependent RNA polymerase elongation complex reveals novel features in motif A dynamics

<正>Dear Editor,Coxsackievirus A16(CV A16)and enterovirus 71(EV71)are currently the two primary causative agents of handfoot-and-mouth disease(HFMD)(Solomon et al.,2010;Mao et al.,2014),threatening health of children worldwide.They both belong to the Enterovirus genus of the     

Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet-platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7A resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin alphaVbeta3 suggests that the Arg binds to the propeller domain, and Asp to the betaA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.     

The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40

The molecular chaperone Hsp40 functions as a dimer. The dimer formation is critical for Hsp40 molecular chaperone activity to facilitate Hsp70 to refold non-native polypeptides. We have determined the crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 that is responsible for Ydj1 dimerization by MAD method. The C-terminal fragment of Ydj1 comprises of the domain III of Ydj1 and the Ydj1 C-terminal dimerization motif. The crystal structure indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. The C terminus of type I Hsp40 Ydj1 from one monomer forms beta-strands with the domain III from the other monomer in the homo-dimer. The L372 from Ydj1 C terminus inserts its side-chain into a hydrophobic pocket on domain III. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers. The domain IIs of Ydj1 monomers that contain the zinc-finger motifs points directly against each other.     

Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA

Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression.     

A wheat HMW glutenin subunit gene reveals a highly repeated structure.

A wheat genomic library was screened with two synthetic oligonucleotides (24 and 25 bases in length) complementary to a partial cDNA clone encoding a glutenin gene [Thompson et al. (1983) Theor. Appl. Genet. 67, 87-96]. Glutenins are large molecular weight aggregated proteins of grain endosperm, and major determinants of bread making quality of wheat. Of the two clones obtained one was fully characterized. It contained the sequence of the high molecular weight subunit of glutenin. The amino acid sequence derived from the gene sequence reveals a mature protein (817 amino acids) with a highly repeated structure of two different motifs corresponding to the high glutamine (35.7%), glycine (20.1%) and proline (13.1%) content. The gene does not contain an intron, and possesses a typical eukaryotic promoter; the RNA initiation site is 25-30 bases downstream.     

Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.     

Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer

BACKGROUND: Utrophin is a large multidomain protein that belongs to a superfamily of actin-binding proteins, which includes dystrophin, alpha-actinin, beta-spectrin, fimbrin, filamin and plectin. All the members of this family contain a common actin-binding region at their N termini and perform a wide variety of roles associated with the actin cytoskeleton. Utrophin is the autosomal homologue of dystrophin, the protein defective in the X-linked Duchenne and Becker muscular dystrophies, and upregulation of utrophin has been suggested as a potential therapy for muscular dystrophy patients. RESULTS: The structure of the actin-binding region of utrophin, consisting of two calponin-homology (CH) domains, has been solved at 3.0 A resolution. It is composed of an antiparallel dimer with each of the monomers being present in an extended dumbell shape and the two CH domains being separated by a long central helix. This extended conformation is in sharp contrast to the compact monomer structure of the N-terminal actin-binding region of fimbrin. CONCLUSIONS: The crystal structure of the actin-binding region of utrophin suggests that these actin-binding domains may be more flexible than was previously thought and that this flexibility may allow domain reorganisation and play a role in the actin-binding mechanism. Thus utrophin could possibly bind to actin in an extended conformation so that the sites previously identified as being important for actin binding may be directly involved in this interaction.     

Crystal structure of the catalytic domain of human atypical protein kinase C-iota reveals interaction mode of phosphorylation site in turn motif

Atypical protein kinases C (aPKCs) play critical roles in signaling pathways that control cell growth, differentiation and survival. Therefore, they constitute attractive targets for the development of novel therapeutics against cancer. The crystal structure of the catalytic domain of atypical PKCiota in complex with the bis(indolyl)maleimide inhibitor BIM1 has been determined at 3.0A resolution within the frame of the European Structural Proteomics Project SPINE. The overall structure exhibits the classical bilobal kinase fold and is in its fully activated form. Both phosphorylation sites (Thr403 in the activation loop, and Thr555 in the turn motif) are well defined in the structure and form intramolecular ionic contacts that make an important contribution in stabilizing the active conformation of the catalytic subunit. The phosphorylation site in the hydrophobic motif of atypical PKCs is replaced by the phosphorylation mimic glutamate and this is also clearly seen in the structure of PKCiota (residue 574). This structure determination for the first time provides the architecture of the turn motif phosphorylation site, which is characteristic for PKCs and PKB/AKT, and is completely different from that in PKA. The bound BIM1 inhibitor blocks the ATP-binding site and puts the kinase domain into an intermediate open conformation. The PKCiota-BIM1 complex is the first kinase domain crystal structure of any atypical PKC and constitutes the basis for rational drug design for selective PKCiota inhibitors.     

Crystal structure of a luteoviral RNA pseudoknot and model for a minimal ribosomal frameshifting motif

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter. Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.     

Crystal structure of yeast tRNAAsp: atomic coordinates

The atomic coordinates of yeast aspartic acid transfer RNA, as determined from a crystallographic investigation to 3 A resolution, are presented. In the ribose phosphate backbone sugars are in the C(3')-endo pucker, except for residues A7, A9, D16, G17, G18, D19, C20, U48, A58, and U60 which are in the C(2')-endo pucker. A least-squares superposition of the phosphorus atoms of yeast tRNAAsp and yeast tRNAPhe enlightens both an overall structural similarity and significant conformational differences. The largest deviations occur in the D-loop and the anticodon region.     

Crystal structure of the yeast metacaspase yca1

Yca1, the only metacaspase in Saccharomyces cerevisiae, is thought to be a clan CD cysteine protease that includes the caspase subfamily. Although yeast is a single cell eukaryote, it can undergo a cell death process reminiscent of apoptosis. Yca1 has been reported to play an important role in the regulation of such apoptotic process. However, the structure and functional mechanism of Yca1 remain largely enigmatic. In this study, we report the crystal structure of the Yca1 metacaspase at 1.7 Å resolution, confirming a caspase-like fold. In sharp contrast to canonical caspases, however, Yca1 exists as a monomer both in solution and in the crystals. Canonical caspase contains six β-strands, with strand β6 pairing up with β6 of another caspase molecule to form a homodimerization interface. In Yca1, an extra pair of antiparallel β-strands forms a continuous β-sheet with the six caspase-common β-strands, blocking potential dimerization. Yca1 was reported to undergo autocatalytic processing in yeast; overexpression in bacteria also led to autoprocessing of Yca1 into two fragments. Unexpectedly, we found that both the autocatalytic processing and the proteolytic activity of Yca1 are greatly facilitated by the presence of calcium (Ca²⁺), but not other divalent cations. Our structural and biochemical characterization identifies Yca1 as a Ca²⁺-activated cysteine protease that may cleave specific substrates during stress response in yeast.