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C D Dickinson

Publications and source records attributed to C D Dickinson.

At least 19 recordsLinked to original sources
PubMed2000

Exosite interactions determine the affinity of factor X for the extrinsic Xase complex.

The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X. This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.

4-Aminobenzoic Acid↗
PubMed1999

Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking.

The upstream coagulation enzymes are homologous trypsin-like serine proteases that typically function in enzyme-cofactor complexes, exemplified by coagulation factor VIIa (VIIa), which is allosterically activated upon binding to its cell surface receptor tissue factor (TF). TF cooperates with VIIa to create a bimolecular recognition surface that serves as an exosite for factor X binding. This study analyzes to what extent scissile bond docking to the catalytic cleft contributes to macromolecular substrate affinity. Mutation of the P1 Arg residue in factor X to Gln prevented activation by the TF.VIIa complex but did not reduce macromolecular substrate affinity for TF.VIIa. Similarly, mutations of the S and S' subsites in the catalytic cleft of the enzyme VIIa failed to reduce affinity for factor X, although the affinity for small chromogenic substrates and the efficiency of factor X scissile bond cleavage were reduced. Thus, docking of the activation peptide bond to the catalytic cleft of this enzyme-cofactor complex does not significantly contribute to affinity for macromolecular substrate. Rather, it appears that the creation of an extended macromolecular substrate recognition surface involving enzyme and cofactor is utilized to generate substrate specificity between the highly homologous, regulatory proteases of the coagulation cascade.

Binding, Competitive↗
PubMed1999

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site.

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.

Antibodies, Monoclonal↗
PubMed1999

Importance of factor VIIa Gla-domain residue Arg-36 for recognition of the macromolecular substrate factor X Gla-domain.

Macromolecular substrate docking with coagulation enzyme-cofactor complexes involves multiple contacts distant from the enzyme's catalytic cleft. Here we characterize the binding of the Gla-domain of macromolecular substrate coagulation factor X to the complex of tissue factor (TF) and VIIa. Site-directed mutagenesis of charged residue side chains in the VIIa Gla-domain identified Arg-36 as being important for macromolecular substrate docking. Ala substitution for Arg-36 resulted in an increased KM and a decreased rate of X activation. X with a truncated Gla-domain was activated by mutant and wild-type VIIa at indistinguishable rates, demonstrating that Arg-36 interactions require a properly folded Gla-domain of the macromolecular substrate. VIIa Arg-36 was also required for effective docking of the X Gla-domain in the absence of phospholipid, demonstrating that the Gla-domain of VIIa participates in protein-protein interactions with X. In the absence of TF, the mutant VIIa had essentially normal function, indicating that the cofactor positions VIIa's Gla-domain for optimal macromolecular substrate docking. Computational docking suggests multiple charge complementary contacts of the X Gla-domain with TF.VIIa. A prominent interaction is made by the functionally important X residue Gla-14 with the center of the extended docking site created by residues in the carboxyl module of TF and the contiguous VIIa Gla-domain. These data demonstrate the functional importance of interactions of the Gla-domains of enzyme and substrate, and begin to elucidate the molecular details of the ternary TF.VIIa.X complex.

1-Carboxyglutamic Acid↗
PubMed1998

Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa.

The catalytic activity of the trypsin-like serine protease coagulation factor VIIa is allosterically regulated. In this work, we employed monoclonal antibodies as probes to analyze conformational changes in the VII protease domain that are induced by zymogen activation, cofactor tissue factor (TF) binding, and active site occupancy. The epitopes of three monoclonal antibodies were mapped using a panel of 57 individual alanine replacement mutants in the protease domain. Two of the antibodies had typical "hot spot" epitopes in a basic cluster above the active site cleft and antibody binding to these epitopes was not affected by zymogen activation, TF binding, or active site occupancy. In contrast, the binding kinetics of VII/VIIa to a monoclonal antibody that mapped to an extended epitope overlapping with the macromolecular substrate exosite was affected by each of the conformational transitions of the VIIa protease domain. The changes in antibody affinity are consistent with a transition from zymogen VII to the TF.VIIa complex, with free enzyme VIIa as an intermediate that retains some zymogen-like features responsible for its low catalytic activity. In contrast, active site occupancy resulted in effects that were qualitatively different from the effects of zymogen activation on the antibody epitope. This provides novel insight into the conformational interdependence between the active site, the region for macromolecular substrate recognition, and the cofactor binding exosite of this allosterically regulated serine protease.

Allosteric Regulation↗
PubMed1998

Allosteric regulation of the cofactor-dependent serine protease coagulation factor VIIa.

The integration of structure and function analysis of the tissue factor-factor VIIa complex has provided a detailed view of the functional surface of the extrinsic activation complex. An incomplete zymogen to enzyme transition is responsible for the strict cofactor dependence of catalytic function of factor VIIa. The mutational analysis demonstrates that factor VIIa is allosterically regulated by specific conformational linkages that involve the cofactor binding site, the catalytic cleft, and the macromolecular substrate exosite. Regions of the flexible activation domain appear to play an important role in the allosteric regulation of this cofactor-dependent coagulation serine protease.

Journal Article↗
PubMed1997

Active site modification of factor VIIa affects interactions of the protease domain with tissue factor.

In the initiation of coagulation, tissue factor (TF) allosterically activates the serine protease factor VIIa (VIIa) through specific interactions with protease domain residues. These interactions, and consequently affinity for TF, may be influenced by conformational changes in the protease domain that result from zymogen-enzyme transition or occupancy of the active site by tight binding inhibitors. In functional competition and direct binding analysis, we determined affinities for zymogen and enzyme species of wild-type VII and of mutants at protease domain residues that contact TF. We demonstrate that TF binding is not influenced by zymogen activation, indicating that the protease domain of zymogen and enzyme dock similarly with TF. In contrast, active site occupancy enhanced the affinity for TF by predominantly decreasing the dissociation rate of the TF.VIIa complex. Of the three interface residues studied, only Met306 played a major role in the inhibitor-induced increase in affinity. Met306 is also important for transmitting the allosteric changes from TF to the active site, resulting in enhanced catalysis. This study thus provides evidence for a bidirectional conformational interdependence of the interface residue Met306 and the active site of VIIa.

Binding Sites↗
PubMed1997

Ca2+ binding to the first epidermal growth factor module of coagulation factor VIIa is important for cofactor interaction and proteolytic function.

Epidermal growth factor-like (EGF) domain Ca2+ binding sites in the homologous coagulation factors VII, IX, and X stabilize the structural orientation of the gamma-carboxyglutamic acid-rich (Gla) domain relative to EGF-1. Site-directed mutagenesis was employed here to analyze the functional importance of Ca2+ binding to EGF-1 in factor VIIa (VIIa), which initiates coagulation in complex with its cofactor, tissue factor (TF). Ala replacements for Asp63 or Gln49 resulted in reduced TF affinity concordant with the number of eliminated Ca2+-coordinating oxygen atoms in the respective side chains. Ca2+ binding to EGF-1 had no major direct effect on contacts with TF residue Gln110 or on interactions of VIIa residues Arg79 and Phe40, suggesting that the stabilized Gla-EGF-1 orientation affects overall docking. Gly, Ala, and Glu replacements at Asp46, which is a Ca2+-coordinating residue at the Gla aromatic stack carboxyl terminus, are consistent with the notion that an increased flexibility of the Gla domain relative to EGF-1 contributes significantly to loss of function. Certain mutants in the EGF-1 Ca2+ site had reduced proteolytic function, suggesting the importance of the high affinity Ca2+ binding site for macromolecular substrate interaction.

1-Carboxyglutamic Acid↗
PubMed1997

An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region.

SATB1 is a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, predominantly expressed in thymocytes. We identified an atypical homeodomain and two Cut-like repeats in SATB1, in addition to the known MAR-binding domain. The isolated MAR-binding domain recognizes a certain DNA sequence context within MARs that is highly potentiated for base unpairing. Unlike the MAR-binding domain, the homeodomain when isolated binds poorly and with low specificity to DNA. However, the combined action of the MAR-binding domain and the homeodomain allows SATB1 to specifically recognize the core unwinding element within the base-unpairing region. The core unwinding element is critical for MAR structure, since point mutations within this core abolish the unwinding propensity of the MAR. The contribution of the homeodomain is abolished by alanine substitutions of arginine 3 and arginine 5 in the N-terminal arm of the homeodomain. Site-directed mutagenesis of the core unwinding element in the 3' MAR of the immunoglobulin heavy chain gene enhancer revealed the sequence 5'-(C/A)TAATA-3' to be essential for the increase in affinity mediated by the homeodomain. SATB1 may regulate T-cell development and function at the level of higher order chromatin structure through the critical DNA structural elements within MARs.

Amino Acid Sequence↗
PubMed1997

The structural basis of function of the TF. VIIa complex in the cellular initiation of coagulation.

Cell surface tissue factor (TF), the major in vivo initiator of coagulation, activates coagulation by binding and allosteric activation of the serine protease factor. VIIa (VIIa). A graphic scheme to account for function of this initial bimolecular activation complex has emerged from the integration of structural with functional analyses. The VIIa light chain, specifically the Gla and EGF-1 domains, form extended hydrophobic contacts with TF which account for most of the free energy of binding. These contacts tether VIIa and facilitate interactions of the protease domain with TF necessary for induction of protease function. Several contact residues in the VIIa protease domain-TF interface are involved in the activation of VIIa by complex allosteric effects. Macromolecular substrate zymogens interact with both the VIIa protease domain and the carboxyl-terminal module of TF. Docking of the VIIa Gla-domain to the latter region of TF appears to contribute to substrate assembly. The current data suggest an extended embrace between TF and VIIa to form the bimolecular enzyme TF.VIIa.

Allosteric Regulation↗
PubMed1996

Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa.

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF-VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF-VIIa with macromolecular substrate.

Binding Sites↗
PubMed1996

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.

Ammonium Sulfate↗
PubMed1994

Crystals of the cell-binding module of fibronectin obtained from a series of recombinant fragments differing in length.

A recombinant fragment corresponding to the cell adhesion module (FNIII10) of human fibronectin has been crystallized at pH 8.6 from solutions containing polyethylene glycol as precipitant. The crystals formed in the space group P2(1) with a = 30.76 A, b = 35.07 A, c = 37.66 A, beta = 106.9 degrees. There is one molecule per asymmetric unit and the crystals diffract beyond 1.75 A resolution. To improve the prospects for successful crystallization of the FNIII10 module, a series of recombinant fragments was produced with minor differences in the length of N or C-terminal segments. Only one of these variants crystallized. Interestingly, the C-terminal residue of this variant formed stable intermolecular contacts with a symmetry-related molecule in the crystal lattice.

Amino Acid Sequence↗
PubMed1994

Crystal structure of the tenth type III cell adhesion module of human fibronectin.

The crystal structure of the cell adhesion module of fibronectin (FNIII10) has been determined at 1.8 A resolution. A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2(1) with a = 30.7, b = 35.1 and c = 37.7 A and beta = 107 degrees. The structure was determined by molecular replacement and refined by least squares methods. The crystallographic R-factor for the final model of the 91 amino acid module plus 56 solvent atoms is 0.18 for 10 to 1.8 A data. The module consists of two layers of beta-sheet, one with three antiparallel strands and the other with four antiparallel strands. The beta-sheets enclose a hydrophobic core of 24 amino acid side-chains. The module contains the RGD cell recognition sequence in a flexible loop connecting two beta-strands. The tertiary structure of the FNIII10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues. A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening.

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.

Amino Acid Sequence↗
PubMed1992

Synthesis and assembly of soybean beta-conglycinin in vitro.

The construction of SP6-derived expression plasmids that encode normal and modified beta-conglycinin subunits is described. With the exception of an additional methionine at their NH2-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids corresponded to mature alpha and beta subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH2-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal alpha subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified beta-conglycinin subunits. The results also show that structural changes at the NH2-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.

Amino Acid Sequence↗
PubMed1991

Slow-growth phenotype of transgenic tomato expressing apoplastic invertase.

The growth of transgenic tomato (Lycopersicon esculentum) plants that express in their apoplast yeast invertase under the control of the cauliflower mosaic virus 35S promoter is severely inhibited. The higher the level of invertase, the greater the inhibition of growth. A second phenotypic characteristic of these transgenic plants is the development of yellow and necrotic spots on the leaves, and leaf curling. Again the severity of the symptoms is correlated with the level of invertase. These symptoms do not develop in shaded leaves indicating the need for photosynthesis. Keeping the plants in the dark for a prolonged period (24 hours) results in the disappearance of leaf starch from the control plants, but not from the plants with apoplastic invertase. These results are consistent with the interpretation that apoplastic invertase prevents photosynthate export from source leaves and that phloem loading includes an apoplastic step.

Journal Article↗