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K I Varughese

Publications and source records attributed to K I Varughese.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of the von Willebrand factor modulator botrocetin.

The binding of von Willebrand factor (vWF) to the platelet receptor, glycoprotein (GP) Ib-IX-V complex, has a key role in the initiation of thrombus formation and is regulated by interactions with extracellular matrix components under the influence of hemodynamic forces. To a certain extent, these effects can be mimicked in vitro by two nonphysiologic modulators, ristocetin and botrocetin. The latter, isolated from the venom of the snake Bothrops jararaca, is a 31-kDa heterodimeric protein that forms a soluble complex with vWF. As an initial step toward understanding the mechanisms that regulate vWF function, we have solved the crystal structure of botrocetin at 1.8 A resolution. Botrocetin exhibits homology with other snake proteins, but contains only one metal binding site as compared to two in Factor IX binding protein and Factor IX/X binding protein and none in flavocetin. A distinctive feature of botrocetin is the presence of a negatively charged surface that may play a role in the association with the vWF A1 domain.

Amino Acid Sequence↗
PubMed2000

A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction.

BACKGROUND: Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS: Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS: The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.

Bacillus subtilis↗
PubMed2000

Modeling and functional analysis of the interaction between von Willebrand factor A1 domain and glycoprotein Ibalpha.

Binding of the von Willebrand factor (vWF) A1 domain to the glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to reactive substrates under high shear stress conditions, a key event in hemostasis and thrombosis. We have now used the known three-dimensional structure of the A1 domain to model the interaction with the GP Ibalpha sequence 271-279, which has previously been implicated in ligand binding. Docking procedures suggested that A1 domain residues in strand beta3 and preceding loop (residues 559-566) as well as in helix alpha3 (residues 594-603) interact with Asp residues 272, 274, 277 and sulfated Tyr residues 278 and 279 in GP Ibalpha. To verify this model, 14 mutant A1 domain fragments containing single or multiple side chain substitutions were tested for their ability to mediate platelet adhesion under flow. Each of the vWF residues Tyr(565), Glu(596), and Lys(599) proved to be strictly required for A1 domain function, which, in agreement with previous findings, was also dependent on Gly(561). Moreover, an accessory functional role was apparent for a group of positively charged residues, including Arg at positions 629, 632, 636 and Lys at positions 643 and 645, possibly acting in concert. There was, however, no evidence from the model that these residues directly participate in forming the complex with GP Ibalpha. These results provide a partial model of the vWF-GP Ibalpha interaction linked to the manifestation of functional activity in platelet adhesion.

Amino Acids↗
PubMed2000

von Willebrand factor conformation and adhesive function is modulated by an internalized water molecule.

Platelet participation in hemostasis and arterial thrombosis requires the binding of glycoprotein (GP) Ibalpha to von Willebrand factor (vWF). Hemodynamic forces enhance this interaction, an effect mimicked by the substitution I546V in the vWF A1 domain. A water molecule becomes internalized near the deleted Ile methyl group. The change in hydrophobicity of the local environment causes positional changes propagated over a distance of 27 A. As a consequence, a major reorientation of a peptide plane occurs in a surface loop involved in GP Ibalpha binding. This distinct vWF conformation shows increased platelet adhesion and provides a structural model for the initial regulation of thrombus formation.

Blood Platelets↗
PubMed1998

Six previously undescribed pyruvate kinase mutations causing enzyme deficiency.

Erythrocyte pyruvate kinase deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. We present 6 previously undescribed mutations of the PKLR gene associated with enzyme deficiency located at cDNA nt 476 G-->T (159Gly-->Val), 884 C-->T (295Ala-->Val), 943 G-->A (315Glu-->Lys), 1022 G-->A (341Gly-->Asp), 1511 G-->T (504Arg-->Leu), and 1528 C-->T (510Arg-->Ter). Two of these mutations are near the substrate binding site: the 315Glu-->Lys (943A) mutation may be involved in Mg2+ binding and 159Gly-->Val (476T) mutation has a possible effect on ADP binding. Four of six mutations produce deduced changes in the shape of the molecule. Two of these mutations, 504Arg-->Leu (1511T) and 510Arg-->Ter (1528T), are located at the interface of domains A and C. One of them (510Arg-->Ter) is a deletion of the C-terminal residues affecting the integrity of the protein. The 504Arg-->Leu mutation eliminates a stabilizing interaction between domains A and C. Changes in amino acid 341(nt 1022) from Gly to Asp cause local perturbations. The mutation 295Ala-->Val (884T) might affect the way pyruvate kinase interacts with other molecules. We review previously described mutations and conclude that there is not yet sufficient data to allow us to draw conclusions regarding genotype/phenotype relationship.

Adenosine Diphosphate↗
PubMed1998

A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site.

Two-component signaling systems are used by bacteria, plants, and lower eukaryotes to adapt to environmental changes. The first component, a protein kinase, responds to a signal by phosphorylating the second component; a response regulator protein that often acts by inducing the expression of specific genes. Response regulators also have an autophosphatase activity that ensures that the proteins are not permanently activated by phosphorylation. The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecular features responsible for this varied autophosphatase activity have not been clearly defined. Using wild-type and mutant derivatives of the sporulation response regulator Spo0F, it has been demonstrated that a key residue in determining the magnitude of this activity is that at position 56 of Spo0F approximately P; this residue is adjacent to the site of phosphorylation, Asp 54. For example, Spo0F approximately P K56N has a 23-fold greater autophosphatase activity (t1/2 = 8 min) than wild-type Spo0F approximately P (t1/2 = 180 min). It is suggested that, by analogy to the GTPase activity of p21(ras) and by examining the crystallographic structure of Spo0F, that the carboxyamide of the mutant Asn 56 may favorably position a catalytic water near the protein acyl phosphate to promote Spo0F approximately P K56N hydrolysis. It is also deduced that Lys 56 in the wild-type protein is critical for the efficient interaction and phosphoryl transfer between Spo0F and it's cognate protein kinase, KinA. Comparison of the known response regulators shows that inefficient autophosphatases (t1/2 on the order of hours) typically contain an amino acid residue with a long side chain at the position equivalent to 56 in Spo0F, whereas efficient autophosphatases (t1/2 on the order of minutes) frequently contain a residue with a carboxyamide or carboxylate side chain at this position. It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match the diverse biological roles played by these proteins.

Adenosine Triphosphate↗
PubMed1998

Formation of a novel four-helix bundle and molecular recognition sites by dimerization of a response regulator phosphotransferase.

A basis for understanding specificity of molecular recognition between phosphorelay proteins has been deduced from the 2.6 A structure of the Spo0B phosphotransferase of the phosphorelay regulating sporulation initiation. Spo0B consists of two domains: an N-terminal alpha-helical hairpin domain and a C-terminal alpha/beta domain. Two subunits of Spo0B dimerize by a parallel association of helical hairpins to form a novel four-helix bundle from which the active histidine protrudes. Docking studies show that both the monomers interact with a Spo0F molecule at the region surrounding the active site aspartate to position it for phosphotransfer. It is apparent that different surfaces of response regulators may be involved in recognition of the protein partners to which they are paired.

Amino Acid Sequence↗
PubMed1998

Crystal structure of the von Willebrand factor A1 domain in complex with the function blocking NMC-4 Fab.

The presence of one or more copies of von Willebrand factor type A domains identifies a superfamily of proteins usually involved in biological processes controlled by specific molecular interactions, often adhesive in nature. We have solved the crystal structure of the prototypic von Willebrand factor A1 domain, essential for the antihemorrhagic activity of platelets, in complex with the function blocking antibody, NMC-4, at 2.2 A resolution. This has led to the recognition of a putative binding groove for the platelet receptor, glycoprotein Ib alpha, formed by two adjacent alpha-helices and a beta-strand. The structure also shows a contact interface between A1 domain pairs, suggesting a hypothetical mechanism for the regulation of protein assembly and heterologous ligand binding mediated by homophilic interactions of type A domains.

Amino Acid Sequence↗
PubMed1997

A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: crystal structure of Spo0F from Bacillus subtilis.

Spo0F is a secondary messenger in the "two-component" system controlling the sporulation of Bacillus subtilis. Spo0F, like the chemotaxis protein CheY, is a single-domain protein homologous to the N-terminal activator domain of the response regulators. We recently reported the crystal structure of a phosphatase-resistant mutant Y13S of Spo0F with Ca2+ bound in the active site. The crystal structure of wild-type Spo0F in the absence of a metal ion is presented here. A comparison of the two structures reveals that the cation induces significant changes in the active site. In the present wild-type structure, the carboxylate of Asp11 points away from the center of the active site, whereas when coordinated to the Ca2+, as in the earlier structure, it points toward the active site. In addition, Asp54, the site of phosphorylation, is blocked by a salt bridge interaction of an Arg side chain from a neighboring molecule. From fluorescence quenching studies with Spo0F Y13W, we found that only the amino acid Arg binds to Spo0F in a saturable manner (Kd = 15 mM). This observation suggests that a small molecule with a shape complementary to the active site and having a guanidinium group might inhibit phosphotransfer between response regulators and their cognate histidine kinases.

Amino Acid Substitution↗
PubMed1997

Crystal structures of the copper and nickel complexes of RNase A: metal-induced interprotein interactions and identification of a novel copper binding motif.

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the alpha-amino group and the N-terminal residues.

Binding Sites↗
PubMed1997

Purification and preliminary crystallographic studies on the sporulation response regulatory phosphotransferase protein, Spo0B, from Bacillus subtilis.

The phosphotransferase protein Spo0B, a component of the sporulation signal transduction system in Bacillus subtilis was expressed from the Escherichia coli strain BL21DE3. It was purified, crystallized, and 2.25 A data measured using the synchrotron source at the Stanford Linear Accelerator Center. The search for heavy atom derivatives is in progress.

Bacillus subtilis↗
PubMed1997

Crystal structure of NMC-4 fab anti-von Willebrand factor A1 domain.

We have solved the crystal structure of the Fab fragment of NMC-4, a mouse monoclonal antibody that binds to the A1 domain of von Willebrand factor (vWF). Two Asp and three Tyr residues in the complementarity determining regions 1 and 3 of the heavy chain exhibited a spatial orientation suggestive of a dominant role in establishing contact with the antigen. A cluster of Asp and Tyr residues occurs also in a region of the platelet glycoprotein (GP) Ib alpha amino terminal domain known to be critically involved in vWF binding. Thus, the structural information obtained with NMC-4 may prove relevant to understand the stereochemical bases of the GP Ib alpha-vWF interaction essential for thrombus formation at sites of vascular lesion.

Amino Acid Sequence↗
PubMed1996

Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray analysis of a Y13S mutant of Spo0F from Bacillus subtilis.

Spo0F, a member of a superfamily of bacterial response regulatory proteins, is crucial to the regulation of sporulation in Bacillus subtilis. As there were difficulties in reproducing crystals of wild-type Spo0F, we report here the crystallization and preliminary studies of a mutant, Y13S protein, which gave well diffracting reproducible crystals. The crystals of the mutant obtained by the hanging-drop method belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2) a = b = 105.1, c = 85.9 A. Diffraction data were collected at 2.8 A at the laboratory source and subsequently 2.05. A data were collected upon flash freezing the crystal at the Stanford Synchrotron Radiation Laboratory. This mutant participates in the phosphorelay in a similar manner to the wild-type protein. The presence of divalent cations are essential for wild-type phosphorylation and the present mutant crystal form is obtained in the presence of calcium.

Journal Article↗
PubMed1996

Altered structural and mechanistic properties of mutant dihydropteridine reductases.

Nine single genetic mutants of rat dihydropteridine reductase (EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position.

Amino Acid Sequence↗
PubMed1995

Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains.

In the molecular scheme of living organisms, adenosine 3',5'-monophosphate (cyclic AMP or cAMP) has been a universal second messenger. In eukaryotic cells, the primary receptors for cAMP are the regulatory subunits of cAMP-dependent protein kinase. The crystal structure of a 1-91 deletion mutant of the type I alpha regulatory subunit was refined to 2.8 A resolution. Each of the two tandem cAMP binding domains provides an extensive network of hydrogen bonds that buries the cyclic phosphate and the ribose between two beta strands that are linked by a short alpha helix. Each adenine base stacks against an aromatic ring that lies outside the beta barrel. This structure provides a molecular basis for understanding how cAMP binds cooperatively to its receptor protein, thus mediating activation of the kinase.

Affinity Labels↗