Crystal structure of the core domain of RhoE/Rnd3: a constitutively activated small G protein

We report the 2.1 A crystal structure of the core G protein domain of the unusual Rho family member RhoE/Rnd3 in complex with endogenous GTP and magnesium. Unlike other small G proteins, RhoE, along with two other proteins Rnd1/Rho6 and Rnd2/RhoN, does not hydrolyze GTP. The main reason for this is the presence of serines in the positions equivalent to Ala59 and Gln61 in Ras. The structure shows that there are still water molecules in similar positions to the waters thought to be involved in the hydrolysis reaction in other G proteins. The structure suggests three not necessarily exclusive explanations for the lack of hydrolysis. The lack of the conserved glutamine raises the energy of the transition state inhibiting hydrolysis. The serines may restrain the waters from moving closer to the GTP, a step that is required to attain the transition state. They also stabilize the GTP-bound conformation of switch II and could prevent conformational changes required during hydrolysis. By superposition of the RhoE structure on structures of Rho family proteins in complex with binding partners, we make predictions on RhoE interactions with these partners.     

Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells

Glucocorticoid hormones stimulate adherens and tight junction formation in Con8 mammary epithelial tumor cells through a multistep process in which the membrane organization of structural apical junction proteins and tight junction sealing is controlled by specific signal transduction components. We have previously shown that dexamethasone stimulation of apical junction formation requires down-regulation of the small GTPase RhoA. Here we identified Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist, as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localized with actin at the cell periphery and induced the localization of the adherens junction protein beta-catenin and the tight junction protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed tight junctions. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organization. Our results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and tight junction sealing.     

Crystal structure of human Edc3 and its functional implications

Edc3 is an enhancer of decapping and serves as a scaffold that aggregates mRNA ribonucleoproteins together for P-body formation. Edc3 forms a network of interactions with the components of the mRNA decapping machinery and has a modular domain architecture consisting of an N-terminal Lsm domain, a central FDF domain, and a C-terminal YjeF-N domain. We have determined the crystal structure of the N-terminally truncated human Edc3 at a resolution of 2.2 Å. The structure reveals that the YjeF-N domain of Edc3 possesses a divergent Rossmann fold topology that forms a dimer, which is supported by sedimentation velocity and sedimentation equilibrium analysis in solution. The dimerization interface of Edc3 is highly conserved in eukaryotes despite the overall low sequence homology across species. Structure-based site-directed mutagenesis revealed dimerization is required for efficient RNA binding, P-body formation, and likely for regulating the yeast Rps28B mRNA as well, suggesting that the dimeric form of Edc3 is a structural and functional unit in mRNA degradation.     

A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3

Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3(Stau) was cloned from the genomic DNA. At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C. botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C. botulinum. The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.     

Crystal structure of zinc-finger domain of Nanos and its functional implications

Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.     

Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.     

Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications

DNA ligases catalyze the crucial step of joining the breaks in duplex DNA during DNA replication, repair and recombination, utilizing either ATP or NAD(+) as a cofactor. Despite the difference in cofactor specificity and limited overall sequence similarity, the two classes of DNA ligase share basically the same catalytic mechanism. In this study, the crystal structure of an NAD(+)-dependent DNA ligase from Thermus filiformis, a 667 residue multidomain protein, has been determined by the multiwavelength anomalous diffraction (MAD) method. It reveals highly modular architecture and a unique circular arrangement of its four distinct domains. It also provides clues for protein flexibility and DNA-binding sites. A model for the multidomain ligase action involving large conformational changes is proposed.     

Crystal structure based design of functional metal/protein hybrids

Preparation of metal/protein hybrids is growing into important topics in the field of bioinorganic chemistry. X-ray crystal structure analyses of them provide direct information on unique interactions of metal cations or metal cofactors to understand and design enzymatic functions. In this mini review, the authors focus on the recent studies on the metal/protein hybrids concerning crystal structure analyses since 2002 and our related works. The precise structural determination promise us to deeply understand coordination chemistry in protein scaffold and shows intriguing suggestions on rational design and application use for biocatalysts, metal drugs and so on.     

Crystal structure of colicin E3: implications for cell entry and ribosome inactivation.

Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 A long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome.     

Crystal structure of Escherichia coli SufA involved in biosynthesis of iron-sulfur clusters: implications for a functional dimer

IscA and SufA are paralogous proteins that play crucial roles in the biosynthesis of Fe-S clusters, perhaps through a mechanism involving transient Fe-S cluster formation. We have determined the crystal structure of E. coli SufA at 2.7A resolution. SufA exists as a homodimer, in contrast to the tetrameric organization of IscA. Furthermore, a C-terminal segment containing two essential cysteine residues (Cys-Gly-Cys), which is disordered in the IscA structure, is clearly visible in one molecule (the alpha1 subunit) of the SufA homodimer. Although this segment is disordered in the other molecule (the alpha2 subunit), computer modeling of this segment based on the well-defined conformation of alpha1 subunit suggests that the four cysteine residues (Cys114 and Cys116 in each subunit) in the Cys-Gly-Cys motif are positioned in close proximity at the dimer interface. The arrangement of these cysteines together with the nearby Glu118 in SufA dimer may allow coordination of an Fe-S cluster and/or an Fe atom.     

Crystal structure of unliganded TRAP: implications for dynamic allostery

Allostery is vital to the function of many proteins. In some cases, rather than a direct steric effect, mutual modulation of ligand binding at spatially separated sites may be achieved through a change in protein dynamics. Thus changes in vibrational modes of the protein, rather than conformational changes, allow different ligand sites to communicate. Evidence for such an effect has been found in TRAP (trp RNA-binding attenuation protein), a regulatory protein found in species of Bacillus. TRAP is part of a feedback system to modulate expression of the trp operon, which carries genes involved in tryptophan synthesis. Negative feedback is thought to depend on binding of tryptophan-bound, but not unbound, TRAP to a specific mRNA leader sequence. We find that, contrary to expectations, at low temperatures TRAP is able to bind RNA in the absence of tryptophan, and that this effect is particularly strong in the case of Bacillus stearothermophilus TRAP. We have solved the crystal structure of this protein with no tryptophan bound, and find that much of the structure shows little deviation from the tryptophan-bound form. These data support the idea that tryptophan may exert its effect on RNA binding by TRAP through dynamic and not structural changes, and that tryptophan binding may be mimicked by low temperature.     

Crystal structure of PTP-SL/PTPBR7 catalytic domain: implications for MAP kinase regulation

Protein tyrosine phosphatases PTP-SL and PTPBR7 are isoforms belonging to cytosolic membrane-associated and to receptor-like PTPs (RPTPs), respectively. They represent a new family of PTPs with a major role in activation and translocation of MAP kinases. Specifically, the complex formation between PTP-SL and ERK2 involves an unusual interaction leading to the phosphorylation of PTP-SL by ERK2 at Thr253 and the inactivating dephosphorylation of ERK2 by PTP-SL. This interaction is strictly dependent upon a kinase interaction motif (KIM) (residues 224-239) situated at the N terminus of the PTP-SL catalytic domain. We report the first crystal structure of the catalytic domain for a member of this family (PTP-SL, residues 254-549, identical with residues 361-656 of PTPBR7), providing an example of an RPTP with single cytoplasmic domain, which is monomeric, having an unhindered catalytic site. In addition to the characteristic PTP-core structure, PTP-SL has an N-terminal helix, possibly orienting the KIM motif upon interaction with the target ERK2. An unusual residue in the catalytically important WPD loop promotes formation of a hydrophobically and electrostatically stabilised clamp. This could induce increased rigidity to the WPD loop and therefore reduced catalytic activity, in agreement with our kinetic measurements. A docking model based on the PTP-SL structure suggests that, in the complex with ERK2, the phosphorylation of PTP-SL should be accomplished first. The subsequent dephosphorylation of ERK2 seems to be possible only if a conformational rearrangement of the two interacting partners takes place.     

Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure

The ROCK-I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin-based focal adhesions. ROCK-I phosphorylates the unconventional G-protein RhoE on multiple N- and C-terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA-induced stress fibre assembly. Here, we provide a molecular explanation for multi-site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK-I kinase domain. RhoE interacts with the C-lobe αG helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK-I catalytic site. Disruption of the ROCK-I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE-mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK-I through protein(s) binding to its effector region. We propose that ROCK-I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling.     

Crystal structure and functional implications of Pyrococcus furiosus hef helicase domain involved in branched DNA processing

DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.     

Crystal structure of YbaK protein from Haemophilus influenzae (HI1434) at 1.8 A resolution: functional implications

Structural genomics of proteins of unknown function most straightforwardly assists with assignment of biochemical activity when the new structure resembles that of proteins whose functions are known. When a new fold is revealed, the universe of known folds is enriched, and once the function is determined by other means, novel structure-function relationships are established. The previously unannotated protein HI1434 from H. influenzae provides a hybrid example of these two paradigms. It is a member of a microbial protein family, labeled in SwissProt as YbaK and ebsC. The crystal structure at 1.8 A resolution reported here reveals a fold that is only remotely related to the C-lectin fold, in particular to endostatin, and thus is not sufficiently similar to imply that YbaK proteins are saccharide binding proteins. However, a crevice that may accommodate a small ligand is evident. The putative binding site contains only one invariant residue, Lys46, which carries a functional group that could play a role in catalysis, indicating that YbaK is probably not an enzyme. Detailed sequence analysis, including a number of newly sequenced microbial organisms, highlights sequence homology to an insertion domain in prolyl-tRNA synthetases (proRS) from prokaryote, a domain whose function is unknown. A HI1434-based model of the insertion domain shows that it should also contain the putative binding site. Being part of a tRNA synthetases, the insertion domain is likely to be involved in oligonucleotide binding, with possible roles in recognition/discrimination or editing of prolyl-tRNA. By analogy, YbaK may also play a role in nucleotide or oligonucleotide binding, the nature of which is yet to be determined.     

Crystal structure of yeast phenylalanine transfer RNA. II. Structural features and functional implications.

The structural features of yeast phenylalanine transfer RNA are analyzed and documented in detail, based on atomic co-ordinates obtained from an extensive crystallographic refinement of the crystal structure of the molecule at 2.7 Å resolution (see preceding paper). We describe here: the relative orientation and the helicity of the base-paired stems; more definitive assignments of tertiary hydrogen bonds involving bases, riboses and phosphates; binding sites for magnesium hydrates, spermine and water; iriter-molecular contacts and base-stacking; flexibility of the molecule; conformational analysis of nucleotides in the structure. Among the more noteworthy features are a considerable irregularity in the helicity of the base-paired stems, a greater flexibility in the anticodon and aminoacyl acceptor arms, and a “coupling” among several conformational angles. The functional implications of these structural features are also discussed.     

Crystal structure of HAb18G/CD147: implications for immunoglobulin superfamily homophilic adhesion

CD147, a member of the immunoglobulin superfamily (IgSF), plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. Importantly, our previous studies have demonstrated that HAb18G/CD147 is a novel hepatocellular carcinoma (HCC)-associated antigen, and HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several matrix metalloproteinases, facilitating invasion and metastasis of HCC cells. In addition, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers. However, the structural basis underlying the multifunctional character of CD147 remains unresolved. We report here the crystal structure of the extracellular portion of HAb18G/CD147 at 2.8A resolution. The structure comprises an N-terminal IgC2 domain and a C-terminal IgI domain, which are connected by a 5-residue flexible linker. This unique C2-I domain organization is distinct from those of other IgSF members. Four homophilic dimers exist in the crystal and adopt C2-C2 and C2-I dimerization rather than V-V dimerization commonly found in other IgSF members. This type of homophilic association thus presents a novel model for homophilic interaction between C2 domains of IgSF members. Moreover, the crystal structure of HAb18G/CD147 provides a good structural explanation for the established multifunction of CD147 mediated by homo/hetero-oligomerizations and should represent a general architecture of other CD147 family members.     

Crystal structure of cbbF from Zymomonas mobilis and its functional implication

A phosphate group at the C1-atom of inositol-monophosphate (IMP) and fructose-1,6-bisphosphate (FBP) is hydrolyzed by a phosphatase IMPase and FBPase in a metal-dependent way, respectively. The two enzymes are almost indiscernible from each other because of their highly similar sequences and structures. Metal ions are bound to residues on the β1- and β2-strands and one mobile loop. However, FBP has another phosphate and FBPases exist as a higher oligomeric state, which may discriminate FBPases from IMPases. There are three genes annotated as FBPases in Zymomonas mobilis, termed also cbbF (ZmcbbF). The revealed crystal structure of one ZmcbbF shows a globular structure formed by five stacked layers. Twenty-five residues in the middle of the sequence form an α-helix and a β-strand, which occupy one side of the catalytic site. A non-polar Leu residue among them is protruded to the active site, pointing out unfavorable access of a bulky charged group to this side. In vitro assays have shown its dimeric form in solution. Interestingly, two β-strands of β1 and β2 are disordered in the ZmcbbF structure. These data indicate that ZmcbbF might structurally belong to IMPase, and imply that its active site would be reorganized in a yet unreported way.     

Crystal structure of Bacillus subtilis YckF: structural and functional evolution

The crystal structure of the YckF protein from Bacillus subtilis was determined with MAD phasing and refined at 1.95A resolution. YckF forms a tight tetramer both in crystals and in solution. Conservation of such oligomerization in other phosphate sugar isomerases indicates that the crystallographically observed tetramer is physiologically relevant. The structure of YckF was compared to with its ortholog from Methanococcus jannaschii, MJ1247. Both of these proteins have phosphate hexulose isomerase activity, although neither of the organisms can utilize methane or methanol as source of energy and/or carbon. Extensive sequence and structural similarities with MJ1247 and with the isomerase domain of glucosamine-6-phosphate synthase from Escherichia coli allowed us to group residues contributing to substrate binding or catalysis. Few notable differences among these structures suggest possible cooperativity of the four active sites of the tetramer. Phylogenetic relationships between obligatory and facultative methylotrophs along with B. subtilis and E. coli provide clues about the possible evolution of genes as they loose their physiological importance.