A. Tulinsky

The Na+ Binding Site of Thrombin

Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow → fast transition is induced by Na+ binding to a site contained within a cylindrical cavity formed by three antiparallel β-strands of the B-chain (Met-Tyr, Lys-Tyr, and Val-Gly) diagonally crossed by the Glu-Glu strand. The site is shaped further by the loop connecting the last two β-strands and is located more than 15 Å away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp of the primary specificity site near its midpoint. The bound Na+ is coordinated octahedrally by the carbonyl oxygen atoms of Tyr, Arg, and Lys, and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na+ binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na+ binding loop is also involved in direct recognition of protein C and antithrombin.

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.

An Ambiguous Structure of a DNA 15-mer Thrombin Complex

The structure of a complex between thrombin and a GGTTGGTGTGGTTGG DNA 15-mer has been analyzed crystallographically. The solution NMR structure of the 15-mer has two stacked G-quartets similar to that found in the previous X-ray structure determination of the 15-mer-thrombin complex [Padmanabhan, Padmanabhan, Ferrara, Sadler & Tulinsky (1993). J. Biol. Chem. 268, 17651-17654]; the strand polarity, however, is reversed from that of the crystallographic structure. The structure of the complex here has been redetermined with better diffraction data confirming the previous crystallographic structure but also indicating that the NMR solution structure fits equally well. Both 15-mer complex structures refined to an R value of about 0.16 presenting a disconcerting ambiguity. Since the two 15-mer structures associate with thrombin in different ways (through the TGT loop in the X-ray and TT loop in the NMR model), other independent lines of physical or chemical evidence are required to resolve the ambiguity.

The molecular environment of the Na+ binding site of thrombin.

When Na+ binds to thrombin, a conformational change is induced that renders the enzyme kinetically faster and more specific in the activation of fibrinogen. Two Na+ binding sites have here been identified crystallographically by exchanging Na+ with Rb+. One is intermolecular, found on the surface between two symmetry-related thrombin molecules. Since it is not present in thrombin crystal structures having different crystal systems, the other Na+ site is the functionally relevant one. The second site has octahedral coordination with the carbonyl oxygen atoms of Arg221A and Lys224 and four conserved water molecules. It is located near Asp189 of the S1 specificity site in an elongated solvent channel (8 x 18 A) formed by four antiparallel beta-strands between Cys182-Cys191 and Val213-Tyr228. This channel, extending from the active site to the opposite surface of the enzyme, was first noted in the hirudin-thrombin structure and contains about 20 conserved water molecules linked together by a hydrogen bonding network that connects to the main chain of thrombin. Although the antiparallel beta-strand interactions of the functional Na+ binding site are the same in prethrombin2, the loops between the strands are very different, so that Asp189 and Arg221A are not positioned properly for either substrate or Na+ binding in prethrombin2. A water molecule with octahedral coordination has also been identified in factor Xa at the topologically equivalent Na+ site position of thrombin. Since activated protein C shows enhanced activity with monovalant cation binding, the same position is probably utilized by Na+. Since thrombin crystals could not be grown in the absence of Na+, the cation was leached from Na(+)-bound thrombin crystals by diffusion/exchange. Although both Na+ and their coordinating water molecules were removed from the Na+ binding sites, the remainder of the thrombin structure was, unexpectedly, the same. The lack of an allosteric change is most likely attributable to crystal packing effects. Thus, the structure of the slow form remains to be established crystallographically.

Crystal and molecular structure of anthracene and biphenylene pillared cofacial diporphyrins

Etude des 2 complexes dinickel (II) anthracene diporphyrine et dicuivre (II) biphenylene diporphyrine

Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase at 2.8 Å resolution

Abstract The structure of 2-keto-3-deoxy-6-phosphogluconate aldolase has been extended to 2.8 A resolution from 3.5 A resolution by multiple isomorphous replacement methods using three heavy-atom derivatives and anomalous Bijvoet differences to 6 A resolution (〈 m 〉 = 0.72). The replacement phases were improved and refined by electron density modification procedures coupled with inverse transform phase angle calculations. A Kendrew model of the molecule was built, which contained all 225 residues of a recently determined amino acid sequence, whereas only 173 were accounted for at 3.5 A resolution. The missing residues were found to be part of the interior of the molecule and not simply an appendage. The molecule folds to form an eight-strand α/β-barrel structure strikingly similar to triosephosphate isomerase, the A-domain of pyruvate kinase and Taka amylase. With a knowledge of the sequence, the nature of the interfaces of the two kinds of crystallographic trimers have been examined, from which it was concluded that the choice of trimers selected in the 3.5 A resolution work was probably correct for trimers in solution. The active site region has been established from the position of the Schiff base forming Lys144 but it has not been possible to confirm it conclusively in independent derivative experiments. An apparent anomaly exists in the location of Glu56 (about 25 A from Lys144). The latter has been reported to assist in catalysis.

Proton magnetic resonance study of lysine-binding to the Kringle 4 domain of human plasminogen: the structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.

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.

The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin.

Angiogenesis inhibitors have gained much public attention recently as anti-cancer agents and several are currently in clinical trials, including angiostatin (Phase I, Thomas Jefferson University Hospital, Philadelphia, PA). We report here the bowl-shaped structure of angiostatin kringles 1-3, the first multi-kringle structure to be determined. All three kringle lysine-binding sites contain a bound bicine molecule of crystallization while the former of kringle 2 and kringle 3 are cofacial. Moreover, the separation of the kringle 2 and kringle 3 lysiner binding sites is sufficient to accommodate the alpha-helix of the 30 residue peptide VEK-30 found in the kringle 2/VEK-30 complex. Together the three kringles produce a central cavity suggestive of a unique domain where they may function in concert.

Localization of the Thrombin-binding Domain on Prothrombin Fragment 2

Co-crystallographic studies have shown that the interaction of human prothrombin fragment 2 (F2) with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1–116), rF2-(55–116), and sF2-(63–116), all of which contain the kringle inner loop (residues 64–93) and the acidic COOH-terminal connecting peptide (residues 94–116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2-(63–90), or the COOH-terminal connecting peptide, sF2-(92–116), do not bind. Thus, contrary to predictions from the crystal structure, the COOH-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63–116) bind saturably to fluorescently labeled active site-blocked thrombin with K d values of 4.1 and 51.3 μm, respectively. The affinity of sF2-(63–116) for thrombin increases about 5-fold (K d = 10 μm) when Val at position 78 is substituted with Glu. F2 and sF2-(63–116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin ∼4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2-(63–116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently labeled hirudin54–65 from active site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the active site and/or exosite I. These studies provide further insight into the regions of F2 that evoke functional changes in thrombin.