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A Tulinsky

Publications and source records attributed to A Tulinsky.

At least 73 records · Page 4Linked to original sources
PubMed1992

Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex.

The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finite-difference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.

Amino Acid Sequence↗
PubMed1991

Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-A resolution.

The crystal structure of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the bovine prothrombin kringle 1 (PTK1) structure as a model and refined by restrained least-squares methods to an R factor of 14.2% at 1.9-A resolution. The K4 structure is similar to that of PTK1, and an insertion of one residue at position 59 of the latter has minimal effect on the protein folding. The PGK4 structure is highly stabilized by an internal hydrophobic core and an extensive hydrogen-bonding network. Features new to this kringle include a cis peptide bond at Pro30 and the presence of two alternate, perpendicular, and equally occupied orientations for the Cys75 side chain. The K4 lysine-binding site consists of a hydrophobic trough formed by the Trp62 and Trp72 indole rings, with anionic (Asp55/Asp57) and cationic (Lys35/Arg71) charge pairs at either end. With the adjacent Asp5 and Arg32 residues, these result in triply charged anionic and cationic clusters (pH of crystals at 6.0), which, in addition to the unusually high accessibility of the Trp72 side chain, serve as an obvious marker of the binding site on the K4 surface. A complex intermolecular interaction occurs between the binding sites of symmetry-related molecules involving a highly ordered sulfate anion of solvation in which the Arg32 side chain of a neighboring kringle occupies the binding site.

Amino Acid Sequence↗
PubMed1991

The refined structure of the epsilon-aminocaproic acid complex of human plasminogen kringle 4.

The crystallographic structure of the plasminogen kringle 4-epsilon-aminocaproic acid (ACA) complex (K4-ACA) has been solved by molecular replacement rotation-translation methods utilizing the refined apo-K4 structure as a search model (Mulichak et al., 1991), and it has been refined to an R value of 0.148 at 2.25-A resolution. The K4-ACA structure consists of two interkringle residues, the kringle along with the ACA ligand, and 106 water molecules. The lysine-binding site has been confirmed to be a relatively open and shallow depression, lined by aromatic rings of Trp62, Phe64, and Trp72, which provide a highly nonpolar environment between doubly charged anionic and cationic centers formed by Asp55/Asp57 and Lys35/Arg71. A zwitterionic ACA ligand molecule is held by hydrogen-bonded ion pair interactions and van der Waals contacts between the charged centers. The lysine-binding site of apo-K4 and K4-ACA have been compared: the rms differences in main-chain and side-chain positions are 0.25 and 0.69 A, respectively, both practically within error of the determinations. The largest deviations in the binding site are due to different crystal packing interactions. Thus, the lysine-binding site appears to be preformed, and lysine binding does not require conformational changes of the host. The results of NMR studies of lysine binding with K4 are correlated with the structure of K4-ACA and agree well.

Amino Acid Sequence↗
PubMed1991

Structure of the hirugen and hirulog 1 complexes of alpha-thrombin.

The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and 0.163, respectively. The binding of hirugen to thrombin is similar to that of the binding of the C-terminal dodecapeptide of hirudin, including that of the terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of sulfation, increases the binding affinity by an order of magnitude, is involved in an extended hydrogen bonding network utilizing all three sulfato oxygen atoms. The hirugen-thrombin complex is the first thrombin structure determined to have an unobstructed active site; this site is practically identical in positioning of catalytic residues and in its hydrogen bonding pattern with that of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1 occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4 spacer to hirugen is disordered in the structure, as is the 3(10) turn of hirugen. The latter must be related to the simultaneous absence both of sulfation and of the last residue of hirudin (Gln65'). In addition, the autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures. Changes in circular dichroism upon hirugen binding are therefore most likely the result of the flexibility associated with this loop.

Amino Acid Sequence↗
PubMed1991

Refined structure of the hirudin-thrombin complex.

The structure of a recombinant hirudin (variant 2, Lys47) human alpha-thrombin complex has been refined using restrained least-squares methods to a crystallographic R-factor of 0.173. The hirudin structure consists of an N-terminal domain folded into a globular unit and a long 17-peptide C-terminal in an extended chain conformation. The N-terminal domain binds at the active-site of thrombin where Ile1' to Tyr3' penetrates to the catalytic triad. The alpha-amino group of Ile1' of hirudin makes a hydrogen bond with OG of Ser195 of thrombin, the side-chains of Ile1' and Tyr3' occupy the apolar site, Thr2' is at the entrance to, but does not enter, the S1 specificity site and Ile1' to Tyr3' form a parallel beta-strand with Ser214 to Gly219. The latter interaction is antiparallel in all other serine proteinase-protein inhibitor complexes. The extended C-terminal segment of hirudin, which is abundant in acidic residues, makes many electrostatic interactions with the fibrinogen binding exosite while the last five residues are in a 3(10) helical turn residing in a hydrophobic patch on the thrombin surface. The precision of the complementarity displayed by these two molecules produces numerous interactions, which although independently generally weak, together are responsible for the high degree of affinity and specificity. Although hirudin-thrombin and D-Phe-Pro-Arg-chloromethyl ketone-thrombin differ in conformation in the autolysis loop (Lys145 to Gly150), this is most likely due to different crystal packing interactions and changes in circular dichroism between the two are probably due to the inherent flexibility of the loop. An RGD sequence, which is generally known to be involved in cell surface receptor interactions, occurs in thrombin and is associated with a long solvent channel filled with water molecules leading to the surface from the end of the S1 site. However, the RGD triplet does not appear to be able to interact in concert in a surface binding mode.

Amino Acid Sequence↗
PubMed1991

Structure of bovine prothrombin fragment 1 refined at 2.25 A resolution.

The structure of bovine prothrombin fragment 1 has been refined at 2.25 A resolution using high resolution measurements made with the synchrotron beam at CHESS. The synchrotron data were collected photographically by oscillation methods (R-merge = 0.08). These were combined with lower order diffractometer data for refinement purposes. The structure was refined using restrained least-squares methods with the program PROLSQ to a crystallographic R-value of 0.175. The structure includes 105 water molecules with occupancies of greater than 0.6. The first 35 residues (Ala1-Leu35) of the N-terminal gamma-carboxy glutamic acid-domain (Ala1-Cys48) of fragment 1 are disordered as are two carbohydrate chains of Mr approximately 5000; the latter two combine to render 40% of the structure disordered. The folding of the kringle of fragment 1 is related to the close intramolecular contact between the inner loop disulfide groups. Half of the conserved sequence of the kringle forms an inner core surrounding these disulfide groups. The remainder of the sequence conservation is associated with the many turns of the main chain. The Pro95 residue of the kringle has a cis conformation and Tyr74 is ordered in fragment 1, although nuclear magnetic resonance studies indicate that the comparable residue of plasminogen kringle 4 has two positions. Surface accessibility calculations indicate that none of the disulfide groups of fragment 1 is accessible to solvent.

Amino Acid Sequence↗
PubMed1991

Comparison of the crystal structures of a flavodoxin in its three oxidation states at cryogenic temperatures.

The focus of this study has been to determine the conformation of the holoprotein of recombinant flavodoxin from Desulfovibrio vulgaris with the FMN in each of its three oxidation states. The structures of the oxidized state of the wild-type flavodoxin at 2.0 A from D. vulgaris was used as a starting model for refinement. Diffraction experiments were conducted at low temperature (-150 degrees C) in order to maintain the oxidation state of interest throughout the intensity data collection. yellow bipyramids by the standard hanging-drop method from 3.2 M-ammonium sulfate in 0.1 M-Tris-HCl buffer at pH 7.0 with protein concentrations ranging from 0.7% to 0.9%. The reduced states of the crystals were achieved through the addition of sodium dithionite at pH 7.0 for the semiquinone (semi-reduced) and at pH 9.0 for the hydroquinone (fully reduced). Data sets consisting of one at room temperature (oxidized state) and three at low temperature (each oxidation state) were collected on a Nicolet P3F/Xentronics area detector X-ray diffractometer system. The four structures, hydroquinone at 2.25 A resolution and all others at 1.9 A resolution, were refined by the restrained parameter least-squares program PROLSQ. The final crystallographic R-values converged to 0.21 (hydroquinone), 0.20 (semiquinone), 0.20 (oxidized, low temperature), and 0.17 (oxidized, room temperature). The reduced states of flavodoxin show a different conformation of the protein polypeptide chain (Asp61-Gly62) in the vicinity of NH(5) of the isoalloxazine group relative to the oxidized state. However, there are only slight conformational differences between the semiquinone and hydroquinone states. In this report, structural comparisons of the three are made, with particular emphasis on the features that might be related to the difference in temperature of the diffraction data collections and differences in the oxidation state of the FMN.

Amino Acid Sequence↗
PubMed1991

Solution conformations of the gamma-carboxyglutamic acid domain of bovine prothrombin fragment 1, residues 1-65.

Molecular dynamics simulations have been performed (AMBER version 3.1) on solvated residues 1-65 of bovine prothrombin fragment 1 (BF1) by using the 2.8-A resolution crystallographic coordinates as the starting conformation for understanding calcium ion-induced conformational changes that precede experimentally observable phospholipid binding. Simulations were performed on the non-metal-bound crystal structure, the form resulting from addition of eight calcium ions to the 1-65 region of the crystal structure, the form resulting from removal of calcium ions after 107 ps and continuing the simulation, and an isolated hexapeptide loop (residues 18-23). In all cases, the 100-ps time scale seemed adequate to sample an ensemble of solution conformers within a particular region of conformation space. The non-metal-containing BF1 did not unfold appreciably during a 106-ps simulation starting from the crystallographic geometry. The calcium ion-containing structure (Ca-BF1) underwent an interesting conformational reorganization during its evolution from the crystal structure: during the time course of a 107-ps simulation, Ca-BF1 experienced a trans----cis isomerization of the gamma-carboxyglutamic acid-21 (Gla-21)-Pro-22 peptide bond. Removal of the calcium ions from this structure followed by 114 ps of additional molecular dynamics showed significant unfolding relative to the final 20-ps average structure of the 107-ps simulation; however, the Gla-21-Pro-22 peptide bond remained cis. A 265-ps simulation on the termini-protected hexapeptide loop (Cys-18 to Cys-23) containing two calcium ions also did not undergo a trans----cis isomerization. It is believed that the necessary activation energy for the transitional event observed in the Ca-BF1 simulation was largely supplied by global conformational events with a possible assist from relief of intermolecular crystal packing forces. The presence of a Gla preceding Pro-22, the inclusion of Pro-22 in a highly strained loop structure, and the formation of two long-lived salt bridges prior to isomerization may all contribute to this finding.

1-Carboxyglutamic Acid↗
PubMed1990

Biochemical and functional characterization of human tissue-type plasminogen activator variants with mutagenized kringle domains.

The cDNA encoding full-length human tissue-type plasminogen activator (t-PA) and five variant cDNAs, constructed by in vitro site-directed mutagenesis, were cloned and expressed in Chinese hamster ovary cells. The variant cDNAs were designed to increase the fibrin affinity of t-PA by mutagenesis in the kringle domains of specific amino acids which are assumed to constitute the lysine-binding site. These amino acids were replaced with the corresponding amino acids present in kringle 1 of plasminogen, which has a high affinity for lysine analogues. The mutants included: rt-PA-Arg125 with a Pro125----Arg mutation; rt-PA-Arg164,Tyr165 with Ser164,Ser165----Arg,Tyr; rt-PA-Arg125,Arg164,Tyr165 with Pro125,Ser164,Ser165----Arg,Arg,Tyr; rt-PA-Arg213 with Val213----Arg; and rt-PA-Arg252 with Thr252----Arg. Compared to wild-type recombinant t-PA (rt-PA), the catalytic efficiency for plasminogen activation was enhanced 4-fold for rt-PA-Arg125, and 3-fold for rt-PA-Arg252 while stimulation of plasminogen activation by CNBr-digested fibrinogen was comparable to wild-type rt-PA for rt-PA-Arg125 and 2-fold enhanced for rt-PA-Arg252. All rt-PA moieties showed a similar concentration-dependent and nearly quantitative binding to fibrin as well as to lysine-Sepharose and induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma. Equieffective concentrations (causing 50% clot lysis in 2 h) were 0.17 micrograms/ml for rt-PA-Arg125, and 0.31 micrograms/ml for rt-PA-Arg252 as compared to 0.55 micrograms/ml for rt-PA. The initial plasma half-life following intravenous bolus injection of 0.25 mg/kg in hamsters was 1.2-2.6 min, not significantly different from wild-type rt-PA (2.4 min). Continuous infusion over 60 min in hamsters with a 125I-fibrin-labeled pulmonary embolus produced 50% clot lysis over background with a dose of 0.9-1.8 mg/kg, which is not markedly superior to wild-type rt-PA (2.1 mg/kg). It is concluded that these variants, designed to mimic the high affinity fibrin-binding site of plasminogen, are not endowed with a markedly improved thrombolytic potency.

Animals↗
PubMed1990

The structure of a complex of recombinant hirudin and human alpha-thrombin.

The crystallographic structure of a recombinant hirudin-thrombin complex has been solved at 2.3 angstrom (A) resolution. Hirudin consists of an NH2-terminal globular domain and a long (39 A) COOH-terminal extended domain. Residues Ile1 to Tyr3 of hirudin form a parallel beta-strand with Ser214 to Glu217 of thrombin with the nitrogen atom of Ile1 making a hydrogen bond with Ser195 O gamma atom of the catalytic site, but the specificity pocket of thrombin is not involved in the interaction. The COOH-terminal segment makes numerous electrostatic interactions with an anion-binding exosite of thrombin, whereas the last five residues are in a helical loop that forms many hydrophobic contacts. In all, 27 of the 65 residues of hirudin have contacts less than 4.0 A with thrombin (10 ion pairs and 23 hydrogen bonds). Such abundant interactions may account for the high affinity and specificity of hirudin.

Amino Acid Sequence↗
PubMed1990

Structure of the lysine-fibrin binding subsite of human plasminogen kringle 4.

Human plasminogen kringle 4, which crystallizes in the orthorhombic system a = 32.15(2), b = 49.01(2), c = 49.04(3) A, space group P2(1)2(1)2(1), four molecules per unit cell, protein volume fraction 0.62, has been determined at 1.9 A resolution. The structure was solved by rotation-translation methods using the structure of bovine prothrombin kringle 1 as a model and it has been refined at 1.9 A resolution to an R-value of 0.142. The root mean square (rms) deviation between the main-chain atoms of the two kringles is about 0.5 A while that between 31 conserved side chains is a surprisingly large 1.2 A. The structure of the lysine binding subsite of fibrin binding of kringle 4 is approximated well by prothrombin kringle 1 but with some notable exceptions. The latter transform the site from a non-binding kringle to one which recognizes lysine and other omega-amino-carboxylic acids. The binding site of the observed kringle 4 structure is also compared with one that was modelled from the structure of kringle 1 of prothrombin fragment 1 and NMR observations. Arginine residues of the binding site of a neighbouring molecule make ion pairs with aspartic acid residues in the binding site of another molecule in the kringle 4 structure.

Amino Acid Sequence↗
PubMed1989

Structure of Ca2+ prothrombin fragment 1 including the conformation of the Gla domain.

The structure of Ca2+ prothrombin fragment 1 has been solved at 2.8-A resolution by X-ray crystallographic methods. Most of the Gla domain of fragment 1 (residues 1-48), which is high homologous with the N-terminal regions of six other blood proteins, cannot be identified in the electron density map of the apo structure. This is not the case when crystals are grown in the presence of Ca2+ ions where the Gla domain exhibits a well-defined folded structure. The folding of the Gla domain is dominated by secondary structure: (a) 3.0 turns of alpha-helix (25%) and (b) five short beta-strands arranged into two beta-structural units (40%). The Cys18-Cys23 disulfide of the small conserved loop of Gla domains is close to a cluster of conserved aromatic residues. The resulting interaction is probably responsible for the fluorescence quenching event accompanying Ca2+ ion binding. Since the Gla domain approximates a discoid, all the Gla residues are easily accessible to solvent. The arrangement of the paired Gla residues (7-8, 20-21, 26-27) is highly suggestive in that they essentially line one edge of the Gla domain creating a potentially intense electronegative environment. This region might well be that associated with phospholipid binding. The kringle structure of Ca2+ fragment 1 is essentially indistinguishable from that of the apoprotein at this stage.

Amino Acid Sequence↗
PubMed1989

Human D-Phe-Pro-Arg-CH2-alpha-thrombin crystallization and diffraction data.

Human alpha-thrombin, inhibited with the high-affinity irreversible inhibitor D-Phe-Pro-Arg-chloromethylketone, has been crystallized from polyethylene glycol 8000 solutions buffered with 0.1 M-sodium phosphate. The crystals are: orthorhombic, a = 67.9(1) A, b = 87.9(1) A, c = 61.0(1) A, space group P2(1)2(1)2(1) with four molecules per unit cell. This gives a protein fraction of 58% consistent with the excellent X-ray diffraction quality of the crystals. A mercury heavy-atom derivative is being prepared from a thioester analogue of D-Phe-Pro-Arg-CH2-alpha-thrombin in anticipation of a complete crystallographic structure determination.

Amino Acid Chloromethyl Ketones↗
PubMed1989

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms.

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.

Crystallization↗
PubMed1988

Crystallization and preliminary diffraction data of Escherichia coli ADP glucose pyrophosphorylase.

ADP glucose pyrophosphorylase from Escherichia coli has been crystallized from polyethylene glycol 8000 solutions. The crystals are: orthorhombic, a = 155(2), b = 153(2), c = 174(2) A, space group P2(1)2(1)2(1), four tetrameric molecules/unit cell. This gives a solvent fraction of about 75% consistent with the relatively poor diffraction quality of crystals (5.0-A resolution) and their sensitivity to x-ray exposure damage. Ways of circumventing the former and improving the latter are proposed.

Crystallization↗
PubMed1988

Structure of prothrombin fragment 1 refined at 2.8 A resolution.

The structure of prothrombin fragment 1, solved at 2.8 A resolution (1 A = 0.1 nm) by a combination of multiple and single isomorphous replacement methods utilizing solvent flattening, has been refined by restrained least-squares methods (R = 0.24), solvent not included, using fairly stringent restraints on the molecular geometry and individual thermal parameters. The inner kringle loop possesses significantly lower B-values than the outer loops even though the former also constitutes a surface of the folded kringle structure. This surface forms the Lys sub-site of the fibrin binding site of other kringles. The hydrogen bonding network and ion pair interactions of fragment 1 appear to maintain a compact folded structure among the various loops of the kringle structure. On the other hand, since there is only one hydrogen bond between the kringle and its preceding 30 residues, considerable flexibility is suggested for the Gla-domain consistent with its disorder in crystals. A chitobiose has been located at the Asn77 glycosylation site, but only a single N-acetyl-glucosamine is ordered at Asn101. The lysine binding site region of other kringles is not properly developed in fragment 1, accounting for its lack of Lys/fibrin affinity. Most of the conserved sequence among 11 different kringles is associated with either: (1) protecting the inner loop disulfides Cys87-127, Cys115-139 upon which the folding is based; or (2) a requirement of the lysine binding site. The remainder of the conservation is generally associated with the ten reverse turns of the folding; of these 40 residues, or about half the sequence, 14 are conserved among eight different turns. The intermolecular packing consists of infinite helical columns of fragment 1 molecules related by a crystallographic 4(3) screw axis, which are held together by van der Waals' interactions of aromatic clusters from different molecules related by a crystallographic 2-fold rotation axis.

Amino Acid Sequence↗
PubMed1988

Preliminary x-ray investigation of an orthorhombic crystal of hevein.

Hevein, a small protein from the latex of Hevea brasiliensis, has been crystallized by the vapor diffusion method using 2-methyl-2,4-pentanediol and CaCl2 as the precipitant agents. The crystals are orthorhombic space group P21212 with a = 21.88, b = 31.90, and c = 51.24 A and one molecule in the asymmetric unit. The crystals are quite stable to x-rays and suitable for a high resolution three-dimensional structure determination.

Antimicrobial Cationic Peptides↗