Biomedical subjects

T J Rydel

Publications and source records attributed to T J Rydel.

9 recordsLinked to original sources

PubMed1992

Structure of a pepsin/renin inhibitor complex reveals a novel crystal packing induced by minor chemical alterations in the inhibitor.

The structure determination by molecular replacement methods of a monoclinic pepsin/renin inhibitor complex crystal, with two molecules in the asymmetric unit, is presented. The atomic model, consisting of two liganded pepsin molecules and 110 water molecules, has been refined to a final crystallographic R value of 0.139 for data between 8 and 2.9 A resolution. The structure reveals a previously undescribed pepsin dimer formed predominantly by polar interactions. Inhibitor binding induces global structural changes in the native enzyme similar, but not identical, to the ones observed in other chemically similar pepsin/renin inhibitor complexes crystallized in an orthorhombic form. A region of the polypeptide chain (residues 292-297) which was not visible in the orthorhombic crystal is well ordered in the presently described structure; possibly induced by crystal contacts. The crystal packing of native pepsin is compared with the two different crystal forms of the inhibited enzyme.

Binding Sites↗

PubMed1992

Peptide mimetics of the thrombin-bound structure of fibrinopeptide A.

Recent work has suggested that the thrombin-bound conformation of fibrinopeptide A exhibits a strand-turn-strand motif, with a beta-turn centered at residues Glu-11 and Gly-12. Our molecular modeling analysis indicates that the published fibrinopeptide conformation cannot bind reasonably to thrombin but that reorientation of two residues by alignment with bovine pancreatic trypsin inhibitor provides a good fit within the deep thrombin cleft and satisfies all of the experimental nuclear Overhauser effect data. Based on this analysis, we have successfully designed and synthesized hybrid peptide mimetic substrates and inhibitors that mimic the proposed beta-turn structure. The results indicate that the turn conformation is an important aspect of thrombin specificity and that our turn mimetic design successfully mimics the thrombin-bound conformation of fibrinopeptide.

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

Inhibitor binding induces structural changes in porcine pepsin.

The refined structures of two isomorphous pepsin/inhibitor complexes demonstrate that significant conformational changes take place upon ligand binding for a mammalian representative of the aspartic proteinase family. These differences can be attributed mostly to the concerted rigid body movements of two separate clusters of residues relative to a central core. One cluster in the amino domain comprises the flap, the adjacent beta strand (sheet IV) and helices, as well as the interconnecting loops. The other, larger cluster is in the carboxy end and corresponds approximately to the flexible subdomain described previously. Similar conformational changes are proposed to occur in renin and cathepsin D.

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

Revised 2.3 A structure of porcine pepsin: evidence for a flexible subdomain.

A revised three-dimensional crystal structure of ethanol-inhibited porcine pepsin refined to an R-factor of 0.171 at 2.3 A resolution is presented and compared to the refined structures of the fungal aspartic proteinases: penicillopepsin, rhizopuspepsin, and endothiapepsin. Pepsin is composed of two nearly equal N and C domains related by an intra dyad. The overall polypeptide fold and active site structures are homologous for pepsin and the fungal enzymes. The weak inhibition of pepsin by ethanol can be explained by the presence of one or more ethanol molecules, in the vicinity of the active site carboxylates, which slightly alter the hydrogen-bonding network and which may compete with substrate binding in the active site. Structural superposition analysis showed that the N domains aligned better than the C-domains for pepsin and the fungal aspartic proteinases: 107-140 C alpha pairs aligned to 0.72-0.85 A rms for the N domains; 64-95 C alpha pairs aligned to 0.78-1.03 A rms for the C domains. The major structural difference between pepsin and the fungal enzymes concerns a newly described subdomain whose conformation varies markedly among these enzyme structures. The subdomain in pepsin comprises nearly 100 residues and is composed of two contiguous segments within the C domain (residues 192-212 and 223-299). the subdomain is connected, or "hinged," to a mixed beta-sheet that forms one of the structurally invariant, active site psi-loops. Relative subdomain displacements as large as a 21.0 degrees rotation and a 5.9 A translation were observed among the different enzymes. There is some suggestion in pepsin that the subdomain may be flexible and perhaps plays a structural role in mediating substrate binding, determining the substrate specificity, or in the activation of the zymogen.

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↗

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↗

PubMed1985

The structure of prothrombin fragment 1 at 3.5-A resolution.

The structure of prothrombin fragment 1 has been determined at 3.5-A resolution by multiple isomorphous replacement methods with four heavy atom derivatives. The final average figure of merit is 0.72. There is a large cylindrical solvent region with an average diameter of 35-40 A along the entire length of the c axis (85 A) centered at about x = y = 1/2. The connected density forming the wall of this channel is not of sufficient extent to account for the 156 residues of fragment 1 and the two accompanying carbohydrate chains totaling 5000 in molecular weight. Deglycosylated fragment 1 crystallizes isomorphously with fragment 1, and a difference map between the two revealed that the sugar chains are severely disordered and reside in the solvent channel. Although the disordered carbohydrate and the complexity of five disulfides in a 126-residue sequence have hampered the complete tracing of the peptide chain, two-thirds of the molecule has been accounted for in the form of an unusually oblate ellipsoid of about 15 X 30 X 35 A. The folding of the molecule has little secondary structure (one alpha-helix (7%), 20% beta-structure) in agreement with dichroism measurements and one of the points of carbohydrate attachment is suggested from the deglycosylated difference map.

Amino Acid Sequence↗