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John Kuriyan

Publications and source records attributed to John Kuriyan.

At least 37 records · Page 2Linked to original sources
PubMed2005

Out-of-plane motions in open sliding clamps: molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen.

Sliding clamps are ring-like multimeric proteins that encircle duplex DNA and serve as mobile DNA-bound platforms that are essential for efficient DNA replication and repair. Sliding clamps are placed on DNA by clamp loader complexes, in which the clamp-interacting elements are organized in a right-handed spiral assembly. To understand how the flat, ring-like clamps might interact with the spiral interaction surface of the clamp loader complex, we have performed molecular dynamics simulations of sliding clamps (proliferating cell nuclear antigen from the budding yeast, humans, and an archaeal species) in which we have removed one of the three subunits so as to release the constraint of ring closure. The simulations reveal significant structural fluctuations corresponding to lateral opening and out-of-plane distortions of the clamp, which result principally from bending and twisting of the beta-sheets that span the intermolecular interfaces, with smaller but similar contributions from beta-sheets that span the intramolecular interfaces within each subunit. With the integrity of these beta-sheets intact, the predominant fluctuations seen in the simulations are oscillations between lateral openings and right-handed spirals. The tendency for clamps to adopt a right-handed spiral conformation implies that once opened, the conformation of the clamp can easily match the spiraling of clamp loader subunits, a feature that is intrinsic to the recognition of DNA and subsequent hydrolysis of ATP by the clamp-bound clamp loader complex.

Archaeal Proteins↗
PubMed2005

C2 can do it, too.

In this issue of Cell, report that the C2 domain of the serine/threonine protein kinase Cdelta is a phosphotyrosine binding domain and present the crystal structure of this C2 domain bound to a peptide containing phosphotyrosine. Prior to this work, C2 domains were thought to bind only to phospholipids or to unphosphorylated proteins, and the SH2 and PTB domains were the only signaling domains known to recognize phosphotyrosine. This new role for the C2 domain links phosphotyrosine recognition directly to serine/threonine kinase activity and reveals an unexpected mechanism for crosstalk between distinct signaling pathways.

Animals↗
PubMed2005

DNA polymerase clamp loaders and DNA recognition.

Clamp loaders are heteropentameric ATPase assemblies that load sliding clamps onto DNA and are critical for processive DNA replication. The DNA targets for clamp loading are double-stranded/single-stranded junctions with recessed 3' ends (primer-template junctions). Here, we briefly review the crystal structures of clamp loader complexes and the insights they have provided into the mechanism of the clamp loading process.

Adenosine Triphosphatases↗
PubMed2005

Mapping the interaction of DNA with the Escherichia coli DNA polymerase clamp loader complex.

Sliding clamps are loaded onto DNA by ATP-dependent clamp loader complexes. A recent crystal structure of a clamp loader-clamp complex suggested an unexpected mechanism for DNA recognition, in which the ATPase subunits of the loader spiral around primed DNA. We report the results of fluorescence-based assays that probe the mechanism of the Escherichia coli clamp loader and show that conserved residues clustered within the inner surface of the modeled clamp loader spiral are critical for DNA recognition, DNA-dependent ATPase activity and clamp release. Duplex DNA with a 5'-overhang single-stranded region (corresponding to correctly primed DNA) stimulates clamp release, as does blunt-ended duplex DNA, whereas duplex DNA with a 3' overhang and single-stranded DNA are ineffective. These results provide evidence for the recognition of DNA within an inner chamber formed by the spiral organization of the ATPase domains of the clamp loader.

Base Sequence↗
PubMed2005

SAXS and the working protein.

In this issue of Structure, Davies et al., 2005, present shape reconstructions for the molecular motor p97 using small angle X-ray scattering (SAXS) and offer insights into how ATP consumption is coupled to cyclical domain motions. This work emphasizes the emerging potential of SAXS for visualizing the workings of biological machines in solution.

Adenosine Triphosphatases↗
PubMed2004

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex.

Clamp-loader complexes are heteropentameric AAA+ ATPases that load sliding clamps onto DNA. The structure of the nucleotide-free Escherichia coli clamp loader had been determined previously and led to the proposal that the clamp-loader cycles between an inactive state, in which the ATPase domains form a closed ring, and an active state that opens up to form a "C" shape. The crystal structure was interpreted as being closer to the active state than the inactive state. The crystal structure of a nucleotide-bound eukaryotic clamp loader [replication factor C (RFC)] revealed a different and more tightly packed spiral organization of the ATPase domains, raising questions about the significance of the conformation seen earlier for the bacterial clamp loader. We describe crystal structures of the E. coli clamp-loader complex bound to the ATP analog ATPgammaS (at a resolution of 3.5 A) and ADP (at a resolution of 4.1 A). These structures are similar to that of the nucleotide-free clamp-loader complex. Only two of the three functional ATP-binding sites are occupied by ATPgammaS or ADP in these structures, and the bound nucleotides make no interfacial contacts in the complex. These results, along with data from isothermal titration calorimetry, molecular dynamics simulations, and comparison with the RFC structure, suggest that the more open form of the E. coli clamp loader described earlier and in the present work corresponds to a stable inactive state of the clamp loader in which the ATPase domains are prevented from engaging the clamp in the highly cooperative manner seen in the fully ATP-loaded RFC-clamp structure.

Adenosine Diphosphate↗
PubMed2004

Structural analysis of autoinhibition in the Ras activator Son of sevenless.

The classical model for the activation of the nucleotide exchange factor Son of sevenless (SOS) involves its recruitment to the membrane, where it engages Ras. The recent discovery that Ras*GTP is an allosteric activator of SOS indicated that the regulation of SOS is more complex than originally envisaged. We now present crystallographic and biochemical analyses of a construct of SOS that contains the Dbl homology-pleckstrin homology (DH-PH) and catalytic domains and show that the DH-PH unit blocks the allosteric binding site for Ras and suppresses the activity of SOS. SOS is dependent on Ras binding to the allosteric site for both a lower level of activity, which is a result of Ras*GDP binding, and maximal activity, which requires Ras*GTP. The action of the DH-PH unit gates a reciprocal interaction between Ras and SOS, in which Ras converts SOS from low to high activity forms as Ras*GDP is converted to Ras*GTP by SOS.

Allosteric Site↗
PubMed2004

Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases.

Soluble guanylate cyclases are nitric oxide-responsive signaling proteins in which the nitric oxide sensor is a heme-binding domain of unknown structure that we have termed the heme-NO and oxygen binding (H-NOX) domain. H-NOX domains are also found in bacteria, either as isolated domains, or are fused through a membrane-spanning region to methyl-accepting chemotaxis proteins. We have determined the crystal structure of an oxygen-binding H-NOX domain of one such signaling protein from the obligate anaerobe Thermoanaerobacter tengcongensis at 1.77-angstroms resolution, revealing a protein fold unrelated to known structures. Particularly striking is the structure of the protoporphyrin IX group, which is distorted from planarity to an extent not seen before in protein-bound heme groups. Comparison of the structure of the H-NOX domain in two different crystal forms suggests a mechanism whereby alteration in the degree of distortion of the heme group is coupled to changes on the molecular surface of the H-NOX domain and potentially to changes in intermolecular interactions.

Amino Acid Sequence↗
PubMed2004

Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex.

Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.

Adenosine Triphosphate↗
PubMed2004

Fluorescence measurements on the E.coli DNA polymerase clamp loader: implications for conformational changes during ATP and clamp binding.

Sliding clamps are ring-shaped proteins that tether DNA polymerases to their templates during processive DNA replication. The action of ATP-dependent clamp loader complexes is required to open the circular clamps and to load them onto DNA. The crystal structure of the pentameric clamp loader complex from Escherichia coli (the gamma complex), determined in the absence of nucleotides, revealed a highly asymmetric and extended form of the clamp loader. Consideration of this structure suggested that a compact and more symmetrical inactive form may predominate in solution in the absence of crystal packing forces. This model has the N-terminal domains of the delta and delta' subunits of the clamp loader close to each other in the inactive state, with the clamp loader opening in a crab-claw-like fashion upon ATP-binding. We have used fluorescence resonance energy transfer (FRET) to investigate the structural changes in the E.coli clamp loader complex that result from ATP-binding and interactions between the clamp loader and the beta clamp. FRET measurements using fluorophores placed in the N-terminal domains of the delta and delta' subunits indicate that the distances between these subunits in solution are consistent with the previously crystallized extended form of the clamp loader. Furthermore, the addition of nucleotide and clamp to the labeled clamp loader does not appreciably alter these FRET distances. Our results suggest that the changes that occur in the relative positioning of the delta and delta' subunits when ATP binds to and activates the complex are subtle, and that crab-claw-like movements are not a significant component of the clamp loader mechanism.

Adenosine Triphosphate↗
PubMed2004

Allostery and coupled sequence variation in nuclear hormone receptors.

The analysis of correlated sequence variation in evolutionarily related proteins is beginning to provide useful information regarding allosteric coupling between different functional sites. Such an analysis has been carried out for the nuclear hormone receptors, and the conclusions tested by making mutations that switch the allosteric response to ligands of RXR heterodimers.

Allosteric Regulation↗
PubMed2004

Crystal structure of the chi:psi sub-assembly of the Escherichia coli DNA polymerase clamp-loader complex.

The chi (chi) and psi (psi) subunits of Escherichia coli DNA polymerase III form a heterodimer that is associated with the ATP-dependent clamp-loader machinery. In E. coli, the chi:psi heterodimer serves as a bridge between the clamp-loader complex and the single-stranded DNA-binding protein. We determined the crystal structure of the chi:psi heterodimer at 2.1 A resolution. Although neither chi (147 residues) nor psi (137 residues) bind to nucleotides, the fold of each protein is similar to the folds of mononucleotide-(chi) or dinucleotide-(psi) binding proteins, without marked similarity to the structures of the clamp-loader subunits. Genes encoding chi and psi proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that residues at the chi:psi interface are highly conserved in both proteins, suggesting that the heterodimeric interaction is of functional significance. The conservation of surface-exposed residues is restricted to the interfacial region and to just two other regions in the chi:psi complex. One of the conserved regions was found to be located on chi, distal to the psi interaction region, and we identified this as the binding site for a C-terminal segment of the single-stranded DNA-binding protein. The other region of sequence conservation is localized to an N-terminal segment of psi (26 residues) that is disordered in the crystal structure. We speculate that psi is linked to the clamp-loader complex by this flexible, but conserved, N-terminal segment, and that the chi:psi unit is linked to the single-stranded DNA-binding protein via the distal surface of chi. The base of the clamp-loader complex has an open C-shaped structure, and the shape of the chi:psi complex is suggestive of a loose docking within the crevice formed by the open faces of the delta and delta' subunits of the clamp-loader.

Adenosine Triphosphate↗
PubMed2003

A myristoyl/phosphotyrosine switch regulates c-Abl.

The c-Abl tyrosine kinase is inhibited by mechanisms that are poorly understood. Disruption of these mechanisms in the Bcr-Abl oncoprotein leads to several forms of human leukemia. We found that like Src kinases, c-Abl 1b is activated by phosphotyrosine ligands. Ligand-activated c-Abl is particularly sensitive to the anti-cancer drug STI-571/Gleevec/imatinib (STI-571). The SH2 domain-phosphorylated tail interaction in Src kinases is functionally replaced in c-Abl by an intramolecular engagement of the N-terminal myristoyl modification with the kinase domain. Functional studies coupled with structural analysis define a myristoyl/phosphotyrosine switch in c-Abl that regulates docking and accessibility of the SH2 domain. This mechanism offers an explanation for the observed cellular activation of c-Abl by tyrosine-phosphorylated proteins, the intracellular mobility of c-Abl, and it provides new insights into the mechanism of action of STI-571.

Animals↗
PubMed2003

Structural basis for the autoinhibition of c-Abl tyrosine kinase.

c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks a phosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571) to inhibit the catalytic activity of Abl, but not that of c-Src.

Benzamides↗
PubMed2003

Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS.

Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras.GTP. The crystal structures suggest that Ras.GTP stabilizes the active site of SOS allosterically, and we show that Ras.GTP forms ternary complexes with SOS(cat) in solution and increases significantly the rate of SOS(cat)-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.

Catalytic Domain↗
PubMed2003

Tandem histone folds in the structure of the N-terminal segment of the ras activator Son of Sevenless.

The Ras activator Son of Sevenless (Sos) contains a Cdc25 homology domain, responsible for nucleotide exchange, as well as Dbl/Pleckstrin homology (DH/PH) domains. We have determined the crystal structure of the N-terminal segment of human Sos1 (residues 1-191) and show that it contains two tandem histone folds. While the N-terminal domain is monomeric in solution, its structure is surprisingly similar to that of histone dimers, with both subunits of the histone "dimer" being part of the same peptide chain. One histone fold corresponds to the region of Sos that is clearly similar in sequence to histones (residues 91-191), whereas the other is formed by residues in Sos (1-90) that are unrelated in sequence to histones. Residues that form a contiguous patch on the surface of the histone domain of Sos are conserved from C. elegans to humans, suggesting a potential role for this domain in protein-protein interactions.

Amino Acid Sequence↗
PubMed2003

Nucleotide-induced conformational changes in an isolated Escherichia coli DNA polymerase III clamp loader subunit.

Sliding clamps are loaded onto DNA by ATP-driven clamp loader complexes. The structure of the E. coli clamp loader in a nucleotide-free state has been determined previously. We now report crystal structures of a truncated form of the isolated gamma-ATPase subunit, gamma(1-243), of the E. coli clamp loader, in nucleotide-free and bound forms. The gamma subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked. The binding of either ATPgammaS or ADP, which are shown to bind with equal affinity to gamma(1-243), induces a change in the relative orientation of the two domains such that nucleotides can be accommodated. This change would break one of the gamma:gamma interfaces seen in the empty clamp loader complex, and may represent one step in the activation process.

Adenosine Triphosphatases↗