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Biomedical subjects

Enrico Di Cera

Publications and source records attributed to Enrico Di Cera.

13 recordsLinked to original sources
PubMed2003

Conserved Ser residues, the shutter region, and speciation in serpin evolution.

The suicide inhibitory mechanism of serine protease inhibitors of the serpin superfamily depends heavily on their structural flexibility, which is controlled in large part by the breach and shutter regions of the central Abeta-sheet. We examined codon usage by the highly conserved residues, Ser-53 and Ser-56, of the shutter region and found a TCN-AGY usage dichotomy for Ser-56 that remarkably is linked to the protostome-deuterostome split. Our results suggest that serpin evolution was driven by phylogenetic speciation and not pressure to fulfill new physiologic functions mitigating against coevolution with the family of serine proteases they inhibit.

Codon↗
PubMed2003

Dissecting substrate recognition by thrombin using the inactive mutant S195A.

The catalytically inactive mutant S195A was used to study the interaction of thrombin with substrates under equilibrium conditions. By monitoring changes in intrinsic fluorescence, we measured dissociation constants for a variety of synthetic substrates, PAR peptides and the inhibitor PPACK. The S195A mutant retains the Na(+)-binding properties of the wild type, and substrate binding to the mutant is enhanced by the presence of Na(+). Temperature dependence studies allowed calculation of the thermodynamic parameters of substrate binding at the active site and showed a negligible deltaC(p). Titration of synthetic substrates carrying substitutions at the P1-P3 positions revealed energetics consistent with the specificity hierarchy identified in hydrolysis by the wild type. Titration with PAR peptides, which interact with both the active site and exosite I of thrombin, also showed consistency with the results obtained with the wild type at steady state. These findings demonstrate that inactive mutants of enzymes make it possible to dissect the equilibrium components linked to substrate binding and complement information on the kinetic properties of the wild type.

Algorithms↗
PubMed2003

Thrombin interactions.

After generation from prothrombin, thrombin plays multiple roles in the blood coagulation cascade that are mediated by interaction with a number of physiologic substrates, effectors, and inhibitors. Structural and mutagenesis studies have helped unravel the molecular basis of thrombin interactions in the context of both well-established and emerging new roles of the enzyme. The functional versatility of thrombin owes much to its evolutionary origin and results from structural determinants and mechanisms that can be exploited by pharmacologic intervention.

Allosteric Site↗
PubMed2002

Three-dimensional models of proteases involved in patterning of the Drosophila Embryo. Crucial role of predicted cation binding sites.

Three-dimensional models of the catalytic domains of Nudel (Ndl), Gastrulation Defective (Gd), Snake (Snk), and Easter (Ea), and their complexes with substrate suggest a possible organization of the enzyme cascade controlling the dorsoventral fate of the fruit fly embryo. The models predict that Gd activates Snk, which in turn activates Ea. Gd can be activated either autoproteolytically or by Ndl. The three-dimensional models of each enzyme-substrate complex in the cascade rationalize existing mutagenesis data and the associated phenotypes. The models also predict unanticipated features like a Ca(2+) binding site in Ea and a Na(+) binding site in Ndl and Gd. These binding sites are likely to play a crucial role in vivo as suggested by mutant enzymes introduced into embryos as mRNAs. The mutations in Gd that eliminate Na(+) binding cause an apparent increase in activity, whereas mutations in Ea that abrogate Ca(2+) binding result in complete loss of activity. A mutation in Ea predicted to introduce Na(+) binding results in apparently increased activity with ventralization of the embryo, an effect not observed with wild-type Ea mRNA.

Animals↗
PubMed2002

Crystal structure of the anticoagulant slow form of thrombin.

Using the thrombin mutant R77aA devoid of the site of autoproteolytic degradation at exosite I, we have solved for the first time the structure of thrombin free of any inhibitors and effector molecules and stabilized in the Na(+)-free slow form. The slow form shows subtle differences compared with the currently available structures of the Na(+)-bound fast form that carry inhibitors at the active site or exosite I. The most notable differences are the displacement of Asp-189 in the S1 specificity pocket, a downward shift of the 190-193 strand, a rearrangement of the side chain of Glu-192, and a significant shift in the position of the catalytic Ser-195 that is no longer within H-bonding distance from His-57. The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties.

Anticoagulants↗
PubMed2002

Ser(214) is crucial for substrate binding to serine proteases.

Highly conserved amino acids that form crucial structural elements of the catalytic apparatus can be used to account for the evolutionary history of serine proteases and the cascades into which they are organized. One such evolutionary marker in chymotrypsin-like proteases is Ser(214), located adjacent to the active site and forming part of the primary specificity pocket. Here we report the mutation of Ser(214) in thrombin to Ala, Thr, Cys, Asp, Glu, and Lys. None of the mutants seriously compromises active site catalytic function as measured by the kinetic parameter k(cat). However, the least conservative mutations result in large increases in K(m) because of lower rates of substrate diffusion into the active site. Therefore, the role of Ser(214) is to promote the productive formation of the enzyme-substrate complex. The S214C mutant is catalytically inactive, which suggests that during evolution the TCN-->AGY codon transitions for Ser(214) occurred through Thr intermediates.

Catalysis↗
PubMed2002

The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo.

Administration of the thrombin mutant W215A/E217A (WE), rationally designed for selective activation of the anticoagulant protein C, elicits safe and potent anticoagulant and antithrombotic effects in a baboon model of platelet-dependent thrombosis. The lowest dose of WE tested (0.011 mg/kg bolus) reduced platelet thrombus accumulation by 80% and was at least as effective as the direct administration of 40-fold more (0.45 mg/kg bolus) activated protein C. WE-treated animals showed no detectable hemorrhage or organ failure. No procoagulant activity could be detected for up to 1 week in baboon plasma obtained following WE administration. These results show that engineered thrombin derivatives that selectively activate protein C may represent useful therapeutic agents for the treatment of thrombotic disorders.

Amino Acid Substitution↗
PubMed2002

The thrombin epitope recognizing thrombomodulin is a highly cooperative hot spot in exosite I.

The functional epitope of thrombin recognizing thrombomodulin was mapped using Ala-scanning mutagenesis of 54 residues located around the active site, the Na(+) binding loop, the 186-loop, the autolysis loop, exosite I, and exosite II. The epitope for thrombomodulin binding is shaped as a hot spot in exosite I, centered around the buried ion quartet formed by Arg(67), Lys(70), Glu(77), and Glu(80), and capped by the hydrophobic residues Tyr(76) and Ile(82). The hot spot is a much smaller subset of the structural epitope for thrombomodulin binding recently documented by x-ray crystallography. Interestingly, the contribution of each residue of the epitope to the binding free energy shows no correlation with the change in its accessible surface area upon formation of the thrombin-thrombomodulin complex. Furthermore, residues of the epitope are strongly coupled in the recognition of thrombomodulin, as seen for the interaction of human growth hormone and insulin with their receptors. Finally, the Ala substitution of two negatively charged residues in exosite II, Asp(100) and Asp(178), is found unexpectedly to significantly increase thrombomodulin binding.

Alanine↗
PubMed2002

Substrate recognition drives the evolution of serine proteases.

A method is introduced to identify amino acid residues that dictate the functional diversity acquired during evolution in a protein family. Using over 80 enzymes of the chymotrypsin family, we demonstrate that the general organization of the phylogenetic tree and its functional branch points are fully accounted for by a limited number of residues that cluster around the active site of the protein and define the contact region with the P1-P4 residues of substrate.

Algorithms↗
PubMed2002

Three-dimensional modeling of thrombin-fibrinogen interaction.

Three-dimensional models of thrombin complexed with large fragments of the fibrinogen Aalpha and Bbeta chains are presented. The models are consistent with the results of recent mutagenesis studies of thrombin and with the information available on naturally occurring fibrinogen mutants. Thrombin recognizes fibrinogen with an extended binding surface, key elements of which are Tyr(76) in exosite I, located about 20 A away from the active site, and the aryl binding site located in close proximity to the catalytic triad. A highly conserved aromatic-Pro-aromatic triplet motif is identified in the primed site region of fibrinogen and other natural substrates of thrombin. The role of this triplet, based on the three-dimensional models, is to correctly orient the substrate for optimal bridge binding to exosite I and the active site. The three-dimensional models suggest a possible pattern of recognition by thrombin that applies generally to other natural substrates.

Amino Acid Sequence↗
PubMed2002

Allosteric effects potentiating the release of the second fibrinopeptide A from fibrinogen by thrombin.

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.

Allosteric Site↗
PubMed2002

Evolution of enzyme cascades from embryonic development to blood coagulation.

Recent delineation of the serine protease cascade controlling dorsal-ventral patterning during Drosophila embryogenesis allows this cascade to be compared with those controlling clotting and complement in vertebrates and invertebrates. The identification of discrete markers of serine protease evolution has made it possible to reconstruct the probable chronology of enzyme evolution and to gain new insights into functional linkages among the cascades. Here, it is proposed that a single ancestral developmental/immunity cascade gave rise to the protostome and deuterostome developmental, clotting and complement cascades. Extensive similarities suggest that these cascades were built by adding enzymes from the bottom of the cascade up and from similar macromolecular building blocks.

Animals↗