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J Kuriyan

Publications and source records attributed to J Kuriyan.

At least 19 recordsLinked to original sources
PubMed2005

Molecular dynamics and protein function.

A fundamental appreciation for how biological macromolecules work requires knowledge of structure and dynamics. Molecular dynamics simulations provide powerful tools for the exploration of the conformational energy landscape accessible to these molecules, and the rapid increase in computational power coupled with improvements in methodology makes this an exciting time for the application of simulation to structural biology. In this Perspective we survey two areas, protein folding and enzymatic catalysis, in which simulations have contributed to a general understanding of mechanism. We also describe results for the F(1) ATPase molecular motor and the Src family of signaling proteins as examples of applications of simulations to specific biological systems.

Animals↗
PubMed2001

Clamp loader structure predicts the architecture of DNA polymerase III holoenzyme and RFC.

Recent determinations of the crystal structure of the Escherichia coli gamma complex and delta-beta assembly have shed light on the bacterial clamp loading reaction. In this review, we discuss the structures of delta-beta and the gamma(3)deltadelta' complex and its mechanism of action as a clamp loader of the E. coli beta sliding clamp. We also expand upon the implications of the structural findings to the structure and function of the eukaryotic clamp loader, RFC, and the structure of E. coli DNA polymerase III holoenzyme.

Animals↗
PubMed2001

Crystal structure of the processivity clamp loader gamma (gamma) complex of E. coli DNA polymerase III.

The gamma complex, an AAA+ ATPase, is the bacterial homolog of eukaryotic replication factor C (RFC) that loads the sliding clamp (beta, homologous to PCNA) onto DNA. The 2.7/3.0 A crystal structure of gamma complex reveals a pentameric arrangement of subunits, with stoichiometry delta':gamma(3):delta. The C-terminal domains of the subunits form a circular collar that supports an asymmetric arrangement of the N-terminal ATP binding domains of the gamma motor and the structurally related domains of the delta' stator and the delta wrench. The structure suggests a mechanism by which the gamma complex switches between a closed state, in which the beta-interacting element of delta is hidden by delta', and an open form similar to the crystal structure, in which delta is free to bind to beta.

Binding Sites↗
PubMed2001

Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III.

The dimeric ring-shaped sliding clamp of E. coli DNA polymerase III (beta subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader gamma complex (homolog of eukaryotic Replication Factor C, RFC). The delta subunit of the gamma complex binds to the beta ring and opens it. The crystal structure of a beta:delta complex shows that delta, which is structurally related to the delta' and gamma subunits of the gamma complex, is a molecular wrench that induces or traps a conformational change in beta such that one of its dimer interfaces is destabilized. Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the beta ring opens spontaneously once a dimer interface is perturbed by the delta wrench.

Amino Acid Sequence↗
PubMed2001

Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange.

Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We have previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343). The structure demonstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2. In this study, we have employed site-directed mutagenesis to investigate the functional significance of the conformational changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordinated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras.Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg(2+)- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, we propose that the interactions of Sos with the switch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation.

Amino Acid Substitution↗
PubMed2001

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation.

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.

Amino Acid Substitution↗
PubMed2001

Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity.

Transient receptor potential (TRP) channels modulate calcium levels in eukaryotic cells in response to external signals. A novel transient receptor potential channel has the ability to phosphorylate itself and other proteins on serine and threonine residues. The catalytic domain of this channel kinase has no detectable sequence similarity to classical eukaryotic protein kinases and is essential for channel function. The structure of the kinase domain, reported here, reveals unexpected similarity to eukaryotic protein kinases in the catalytic core as well as to metabolic enzymes with ATP-grasp domains. The inclusion of the channel kinase catalytic domain within the eukaryotic protein kinase superfamily indicates a significantly wider distribution for this group of signaling proteins than suggested previously by sequence comparisons alone.

Amino Acid Motifs↗
PubMed2001

The TGF beta receptor activation process: an inhibitor- to substrate-binding switch.

The type I TGF beta receptor (T beta R-I) is activated by phosphorylation of the GS region, a conserved juxtamembrane segment located just N-terminal to the kinase domain. We have studied the molecular mechanism of receptor activation using a homogeneously tetraphosphorylated form of T beta R-I, prepared using protein semisynthesis. Phosphorylation of the GS region dramatically enhances the specificity of T beta R-I for the critical C-terminal serines of Smad2. In addition, tetraphosphorylated T beta R-I is bound specifically by Smad2 in a phosphorylation-dependent manner and is no longer recognized by the inhibitory protein FKBP12. Thus, phosphorylation activates T beta R-I by switching the GS region from a binding site for an inhibitor into a binding surface for substrate. Our observations suggest that phosphoserine/phosphothreonine-dependent localization is a key feature of the T beta R-I/Smad activation process.

Activin Receptors, Type I↗
PubMed2000

Structural mechanism for STI-571 inhibition of abelson tyrosine kinase.

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.

Animals↗
PubMed2000

Cell science and protein crystal growth research for the International Space Station.

The recent National Research Council report, Future Biotechnology Research on the International Space Station, evaluates NASA's plans for research in cell science and protein crystal growth to be conducted on the International Space Station. This report concludes that the NASA biotechnology programs have the potential to significantly impact relevant scientific fields and to increase understanding and insight into fundamental biological issues. In order to realize the potential impacts, NASA must focus its research programs by selecting specific questions related to gravitational forces' role in cell behavior and by using the microgravity environment as a tool to determine the structure of macromolecules with important biological implications. Given the time and volume constraints associated with space-based experiments, instrumentation to be used on the space station must be designed to maximize the productivity of researchers, and NASA's recruitment of investigators and support for space station experiments should aim to encourage and facilitate cutting-edge research.

Biotechnology↗
PubMed2000

A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases.

Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. We present the crystal structure of the catalytic unit of a primase enzyme, that of a approximately 320 residue fragment of Escherichia coli primase, determined at 2.9 A resolution. Central to the catalytic unit is a TOPRIM domain that is strikingly similar in its structure to that of corresponding domains in DNA topoisomerases, but is unrelated to the catalytic centers of other DNA or RNA polymerases. The catalytic domain of primase is crescent-shaped, and the concave face of the crescent is predicted to accommodate about 10 base-pairs of RNA-DNA duplex in a loose interaction, thereby limiting processivity.

Amino Acid Sequence↗
PubMed2000

Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha.

BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs). The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif. Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor. RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution. Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide. The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues. CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them. The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS. In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region. Versatility in the specific recognition of NLSs relies on the modular.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage.

The protein encoded by gene 45 of T4 bacteriophage (gene 45 protein or gp45), is responsible for tethering the catalytic subunit of T4 DNA Polymerase to DNA during high-speed replication. Also referred to as a sliding DNA clamp, gp45 is similar in its function to the processivity factors of bacterial and eukaryotic DNA polymerases, the beta-clamp and PCNA, respectively. Crystallographic analysis has shown that the beta-clamp and PCNA form highly symmetrical ring-shaped structures through which duplex DNA can be threaded. Gp45 shares no sequence similarity with beta-clamp or PCNA, and sequence comparisons have not been able to establish whether it adopts a similar structure. We have determined the crystal structure of gp45 from T4 bacteriophage at 2.4 A resolution, using multiple isomorphous replacement. The protein forms a trimeric ring-shaped assembly with overall dimensions that are similar to those of the bacterial and eukaryotic processivity factors. Each monomer of gp45 contains two domains that are very similar in chain fold to those of beta-clamp and PCNA. Despite an overall negative charge, the inner surface of the ring is in a region of positive electrostatic potential, consistent with a mechanism in which DNA is threaded through the ring.

Amino Acid Sequence↗
PubMed2000

Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416). Effect of introducing a high affinity intramolecular SH2 ligand.

The Src family tyrosine kinase Hck possesses two phosphorylation sites, Tyr(527) and Tyr(416), that affect the catalytic activity in opposite ways. When phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI. We mutated this sequence to YEEI and show that this mutant form of Hck cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is also involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-Hck mutant in a similar manner to wild-type Hck, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr(416), is the autophosphorylation site in the activation loop. Phosphorylation of Tyr(416) is required for Hck activation. We mutated this residue to alanine and characterized its catalytic activity. The Y416A mutant shows a higher K(m) value for peptide and a lower V(max) than autophosphorylated wild-type Hck. We also present evidence for cross-talk between the activation loop and the intramolecular binding of the SH2 and SH3 domains.

Amino Acid Sequence↗
PubMed1999

Crystal structure of an archaebacterial DNA polymerase.

BACKGROUND: Members of the Pol II family of DNA polymerases are responsible for chromosomal replication in eukaryotes, and carry out highly processive DNA replication when attached to ring-shaped processivity clamps. The sequences of Pol II polymerases are distinct from those of members of the well-studied Pol I family of DNA polymerases. The DNA polymerase from the archaebacterium Desulfurococcus strain Tok (D. Tok Pol) is a member of the Pol II family that retains catalytic activity at elevated temperatures. RESULTS: The crystal structure of D. Tok Pol has been determined at 2.4 A resolution. The architecture of this Pol II type DNA polymerase resembles that of the DNA polymerase from the bacteriophage RB69, with which it shares less than approximately 20% sequence identity. As in RB69, the central catalytic region of the DNA polymerase is located within the 'palm' subdomain and is strikingly similar in structure to the corresponding regions of Pol I type DNA polymerases. The structural scaffold that surrounds the catalytic core in D. Tok Pol is unrelated in structure to that of Pol I type polymerases. The 3'-5' proofreading exonuclease domain of D. Tok Pol resembles the corresponding domains of RB69 Pol and Pol I type DNA polymerases. The exonuclease domain in D. Tok Pol is located in the same position relative to the polymerase domain as seen in RB69, and on the opposite side of the palm subdomain compared to its location in Pol I type polymerases. The N-terminal domain of D. Tok Pol has structural similarity to RNA-binding domains. Sequence alignments suggest that this domain is conserved in the eukaryotic DNA polymerases delta and epsilon. CONCLUSIONS: The structure of D. Tok Pol confirms that the modes of binding of the template and extrusion of newly synthesized duplex DNA are likely to be similar in both Pol II and Pol I type DNA polymerases. However, the mechanism by which the newly synthesized product transits in and out of the proofreading exonuclease domain has to be quite different. The discovery of a domain that seems to be an RNA-binding module raises the possibility that Pol II family members interact with RNA.

Amino Acid Sequence↗