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Biomedical subjects

V C Yee

Publications and source records attributed to V C Yee.

At least 19 recordsLinked to original sources
PubMed2006

Genetic diversity and function in the human cytosolic sulfotransferases.

Amino-acid substitutions, which result from common nonsynonymous (NS) polymorphisms, may dramatically alter the function of the encoded protein. Gaining insight into how these substitutions alter function is a step toward acquiring predictability. In this study, we incorporated gene resequencing, functional genomics, amino-acid characterization and crystal structure analysis for the cytosolic sulfotransferases (SULTs) to attempt to gain predictability regarding the function of variant allozymes. Previously, four SULT genes were resequenced in 118 DNA samples. With additional resequencing of the remaining eight SULT family members in the same DNA samples, a total of 217 polymorphisms were revealed. Of 64 polymorphisms identified within 8785 bp of coding regions from SULT genes examined, 25 were synonymous and 39 were NS. Overall, the proportion of synonymous changes was greater than expected from a random distribution of mutations, suggesting the presence of a selective pressure against amino-acid substitutions. Functional data for common variants of five SULT genes have been previously published. These data, together with the SULT1A1 variant allozyme data presented in this paper, showed that the major mechanism by which amino acid changes altered function in a transient expression system was through decreases in immunoreactive protein rather than changes in enzyme kinetics. Additional insight with regard to mechanisms by which NS single nucleotide polymorphisms alter function was sought by analysis of evolutionary conservation, physicochemical properties of the amino-acid substitutions and crystal structure analysis. Neither individual amino-acid characteristics nor structural models were able to accurately and reliably predict the function of variant allozymes. These results suggest that common amino-acid substitutions may not dramatically alter the protein structure, but affect interactions with the cellular environment that are currently not well understood.

Amino Acid Substitution↗
PubMed2001

Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2.

The leukocyte integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha(M)beta(2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma(383)TMKIIPFNRLTIG(395), P2-C, within the gamma-module of the D-domain of fibrinogen, is a recognition site for alpha(M)beta(2) and alpha(X)beta(2). We have now identified the complementary sequences within the alpha(M)I-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha(M)beta(2), alpha(X)beta(2), and alpha(L)beta(2), i.e. the alpha(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha(M)I-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha(M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.

Amino Acid Sequence↗
PubMed2001

Crystal structure of the human prion protein reveals a mechanism for oligomerization.

The pathogenesis of transmissible encephalopathies is associated with the conversion of the cellular prion protein, PrP(C), into a conformationally altered oligomeric form, PrP(Sc). Here we report the crystal structure of the human prion protein in dimer form at 2 A resolution. The dimer results from the three-dimensional swapping of the C-terminal helix 3 and rearrangement of the disulfide bond. An interchain two-stranded antiparallel beta-sheet is formed at the dimer interface by residues that are located in helix 2 in the monomeric NMR structures. Familial prion disease mutations map to the regions directly involved in helix swapping. This crystal structure suggests that oligomerization through 3D domain-swapping may constitute an important step on the pathway of the PrP(C) --> PrP(Sc) conversion.

Crystallography, X-Ray↗
PubMed2001

Novel Y283C mutation of the A subunit for coagulation factor XIII: molecular modelling predicts its impaired protein folding and dimer formation.

In an Italian patient with severe factor XIII deficiency, a novel mutation, Y283C (TAT to TGT), was identified heterozygously by nucleotide sequencing analysis in exon VII of the gene for the A subunit. The presence of this mutation was confirmed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in the proband and his brother. Molecular modelling predicts that the mutant molecule would be misfolded. It is probable that the impaired folding of the mutant Y283C A subunit led to its instability, which is at least in part responsible for the factor XIII deficiency of this patient.

Child↗
PubMed2001

Crystallization and preliminary X-ray analysis of the 12S central subunit of transcarboxylase from Propionibacterium shermanii.

The hexameric 12S central subunit of transcarboxylase has been crystallized in both free and substrate-bound forms. The apo crystals belong to the cubic space group P4(2)32, with unit-cell parameters a = b = c = 188.5 A, and diffract to 3.5 A resolution. Crystals of two substrate-bound complexes, 12S with methylmalonyl CoA and 12S with malonyl CoA, are isomorphous and belong to space group C2, with unit-cell parameters a = 115.5, b = 201.4, c = 146.9 A, beta = 102.7 degrees. These crystals diffract to 1.9 A resolution with synchrotron radiation. Two useful heavy-atom phasing derivatives of methylmalonyl CoA-bound crystals have been obtained by co-crystallization or crystal soaking.

Acyl Coenzyme A↗
PubMed2000

Interaction of the factor XIII activation peptide with alpha -thrombin. Crystal structure of its enzyme-substrate analog complex.

The serine protease thrombin proteolytically activates blood coagulation factor XIII by cleavage at residue Arg(37); factor XIII in turn cross-links fibrin molecules and gives mechanical stability to the blood clot. The 2.0-A resolution x-ray crystal structure of human alpha-thrombin bound to the factor XIII-(28-37) decapeptide has been determined. This structure reveals the detailed atomic level interactions between the factor XIII activation peptide and thrombin and provides the first high resolution view of this functionally important part of the factor XIII molecule. A comparison of this structure with the crystal structure of fibrinopeptide A complexed with thrombin highlights several important determinants of thrombin substrate interaction. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in thrombin. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both factor XIII-(28-37) and fibrinopeptide A. Finally, the hydrophobic residues, which occupy the aryl binding site of thrombin determine the substrate conformation further away from the catalytic residues. In the case of factor XIII-(28-37), the aryl binding site is shared by hydrophobic residues P4 (Val(34)) and P9 (Val(29)). A bulkier residue in either of these sites may alter the substrate peptide conformation.

Amino Acid Sequence↗
PubMed2000

Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor.

The atrial natriuretic peptide (ANP) hormone is secreted by the heart in response to an increase in blood pressure. ANP exhibits several potent anti-hypertensive actions in the kidney, adrenal gland and vascular system. These actions are induced by hormone binding extracellularly to the ANP receptor, thereby activating its intracellular guanylyl cyclase domain for the production of cyclic GMP. Here we present the crystal structure of the glycosylated dimerized hormone-binding domain of the ANP receptor at 2.0-A resolution. The monomer comprises two interconnected subdomains, each encompassing a central beta-sheet flanked by alpha-helices, and exhibits the type I periplasmic binding protein fold. Dimerization is mediated by the juxtaposition of four parallel helices, arranged two by two, which brings the two protruding carboxy termini into close relative proximity. From affinity labelling and mutagenesis studies, the ANP-binding site maps to the side of the dimer crevice and extends to near the dimer interface. A conserved chloride-binding site is located in the membrane distal domain, and we found that hormone binding is chloride dependent. These studies suggest mechanisms for hormone activation and the allostery of the ANP receptor.

Animals↗
PubMed1999

Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography.

The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331-338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant.

Binding Sites↗
PubMed1998

Structural determinants of the bifunctional corn Hageman factor inhibitor: x-ray crystal structure at 1.95 A resolution.

Corn Hageman factor inhibitor (CHFI) is a bifunctional 127 residue, 13.6 kDa protein isolated from corn seeds. It inhibits mammalian trypsin and Factor XIIa (Hageman Factor) of the contact pathway of coagulation as well as alpha-amylases from several insect species. Among the plasma proteinases, CHFI specifically inhibits Factor XIIa without affecting the activity of other coagulation proteinases. We have isolated CHFI from corn and determined the crystallographic structure at 1.95 A resolution. Additionally, we have solved the structure of the recombinant protein produced in Escherichia coli at 2.2 A resolution. The two proteins are essentially identical. The proteinase binding loop is in the canonical conformation for proteinase inhibitors. In an effort to understand alpha-amylase inhibition by members of the family of 25 cereal trypsin/alpha-amylase inhibitors, we have made three-dimensional models of several proteins in the family based on the CHFI coordinates and the coordinates determined for wheat alpha-amylase inhibitor 0.19 [Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., and Morimoto, T. (1997) Biochemistry 36, 13503-13511]. From an analysis of the models and a structure-based sequence analysis, we propose a testable hypothesis for the regions of these proteins which bind alpha-amylase. In the course of the investigations, we have found that the cereal trypsin/alpha-amylase inhibitor family is evolutionarily related to the family of nonspecific lipid-transfer proteins of plants. This is a new addition to the group which now consists of the trypsin/alpha-amylase inhibitors, 2S seed storage albumins, and the lipid-transfer family. Apparently, the four-helix conformation has been a successful vehicle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer.

Amino Acid Sequence↗
PubMed1998

Identification of a novel recognition sequence for integrin alphaM beta2 within the gamma-chain of fibrinogen.

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

Amino Acid Sequence↗
PubMed1998

Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody.

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

Amino Acid Sequence↗
PubMed1997

The core domain of the tissue transglutaminase Gh hydrolyzes GTP and ATP.

Tissue transglutaminase (TGase II) catalyzes the posttranslational modification of proteins by transamidation of available glutamine residues and is also a guanosinetriphosphatase (GTPase) and adenosinetriphosphatase (ATPase). Based on its homology with factor XIIIA, an extracellular transglutaminase, the structure of TGase II is likely composed of an N-terminal beta-sandwich domain, an alpha/beta catalytic core, and two C-terminally located beta-barrels. Here we used a domain-deletion approach to identify the GTP and ATP hydrolytic domains of TGase II. Full-length TGase II and two domain-deletion mutants, one retaining the N-terminal beta-sandwich and core domains (betaSCore) and the other retaining only the core domain, were expressed as glutathione S-transferase (GST) fusion proteins and purified. GST-Full and GST-betaSCore exhibited calcium-dependent TGase activity, whereas GST-Core had no detectable TGase activity, indicating the beta-sandwich domain is required for TGase activity but the C-terminal beta-barrels are not. All three GST-TGase II fusion proteins were photoaffinity-labeled with [alpha-32P]-8-azidoGTP and were able to bind GTP-agarose. The GTPase activity of GST-betaSCore was equivalent to that of GST-Full, whereas the ATPase activity was approximately 40% higher than GST-Full. GST-Core had approximately 50% higher GTPase activity and approximately 75% higher ATPase activity than GST-Full. The GTPase and ATPase activities of each of the GST-TGase II fusion proteins were inhibited in a dose-dependent manner by both GTPgammaS and ATPgammaS. These results demonstrate that the GTP and ATP hydrolysis sites are localized within the core domain of TGase II and that neither the N-terminal beta-sandwich domain nor the C-terminal beta-barrels are required for either GTP or ATP hydrolysis. Taken together with previous work [Singh, U. S., Erickson, J. W., & Cerione, R. A. (1995) Biochemistry 34, 15863-15871; Lai, T.-S., Slaughter, T. F., Koropchak, C. M., Haroon, Z. A., & Greenberg, C. S. (1996) J. Biol. Chem. 271, 31191-31195] the results of this study indicate that the GTP and ATP hydrolysis sites are localized to a 5. 5 kDa (47 amino acid) region at the start of the core domain.

Adenosine Triphosphatases↗
PubMed1997

Consequences of seven novel mutations on the expression and structure of keratinocyte transglutaminase.

We report the molecular characterization of seven new keratinocyte transglutaminase mutations (R315C, S358R, V379L, G473S, R687C, deletion Delta679-696, R127Stop) found in lamellar ichthyosis patients. Arg-315, Ser-358, Val-379, and Gly-473 are highly conserved residues in transglutaminases while Arg-687 and Delta679-696 are not. All mutations strongly decreased transglutaminase activity and protein levels. The mutation R127Stop diminished the amount of mRNA. Structural analysis of these mutations based on the factor XIII A-subunit crystal structure demonstrated that Arg-315, Ser-358, Val-379, and Gly-473 are located in the catalytic core domain, and Arg-687 and the deletion are in the beta-barrel domains. The side chains of amino acids Arg-315, Ser-358, and Gly-473 make ionic and hydrogen bonds important for folding and structural stability of the enzyme but are not directly involved in catalysis. Val-379 is two amino acids away from the active site cysteine, and its change into leucine disturbs the active site structure. The decreased activity and protein level after expression of the R687C and Delta679-696 TGK cDNA in TGK negative keratinocytes excluded that they are polymorphisms. These results identify important amino acids in the central core domain of transglutaminases and show that the C-terminal end influences the structural and functional integrity of TGK.

Arginine↗
PubMed1997

Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.

BACKGROUND: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. RESULTS: The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. CONCLUSIONS: The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

Amino Acid Sequence↗
PubMed1997

Structural analysis of a missense mutation (Val414Phe) in the catalytic core domain of the factor XIII(A) subunit.

Molecular analysis has been performed on a Malaysian patient with a severe bleeding disorder due to factor XIII(A) subunit deficiency. Total mRNA was isolated from the patient's leucocytes and four overlapping segments corresponding to the entire coding region of the A subunit cDNA were amplified by RT-PCR. The cDNA segments amplified efficiently and were of expected size. Direct sequencing of the complete reading frame revealed a single homozygous base change (nt 1327G-T) in exon 10 corresponding to a missense mutation, Val414Phe, in the catalytic core domain of the A subunit monomer. The mutation eliminates a BsaJ1 restriction site and family screening showed that both parents were heterozygous for the defect. The base substitution was absent in 55 normal individuals. Val414 is a highly conserved residue in the calcium-dependent transglutaminase enzyme family. Computer modelling based on 3D crystallographic data predicts that the bulky aromatic side chain of the substituted phenylalanine residue distorts protein folding and destabilizes the molecule. In addition, conformation changes in the adjacent catalytic and calcium binding regions of the A subunit are likely to impair the enzymatic activity of any protein synthesized.

Adult↗
PubMed1997

Transglutaminase 1 mutations in autosomal recessive congenital ichthyosis: private and recurrent mutations in an isolated population.

Autosomal recessive congenital ichthyosis (ARCI) is a rare, heterogenous keratinization disorder of the skin, classically divided into two clinical subtypes, lamellar ichthyosis (LI) and nonbullous congenital ichthyosiformis erythroderma (CIE). Recently, strong evidence for the involvement of the transglutaminase 1 gene (TGM1) in LI has evolved. We have studied ARCI in the isolated Finnish population, in which recessive disorders are often caused by single mutations enriched by a founder effect. Surprisingly, five different mutations of TGM1 (Arg141His, Arg142Cys, Gly217Ser, Val378Leu, and Arg395Leu) were found in Finnish ARCI patients. In addition to affected LI patients, we also identified TGM1 mutations in CIE patients. Moreover, haplotype analysis of the chromosomes carrying the most common mutation, a C-->T transition changing Arg142 to Cys, revealed that the same mutation has been introduced twice in the Finnish population. In addition to this Arg142Cys mutation, three other mutations, in Arg141 and Arg142, have been described elsewhere, in other populations. These findings suggest that this region of TGM1 is more susceptible to mutation. The corresponding amino acid sequence is conserved in other transglutaminases, but, for example, coagulation factor XIII (FXIII) mutations do not cluster in this region. Protein modeling of the Arg142Cys mutation suggested disruption or destabilization of the protein. In transfection studies, the closely related transglutaminase FXIII protein with the corresponding mutation was shown to be susceptible to degradation in COS cells, further supporting evidence of the destabilizing effect of the Arg142Cys mutation in TGM1.

Adolescent↗