PDBsum entry 1v2p

Hydrolase

PDB id: 1v2p

Contents

Protein chain

223 a.a. *

Ligands

SO4

ANH

Metals

_CA

Waters ×61

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Understanding protein-Ligand interactions: the price of protein flexibility.

Authors

D.Rauh, G.Klebe, M.T.Stubbs.

Ref.

J Mol Biol, 2004, 335, 1325-1341. [DOI no: 10.1016/j.jmb.2003.11.041]

PubMed id

14729347

Abstract

In order to design selective, high-affinity ligands to a target protein, it is advantageous to understand the structural determinants for protein-ligand complex formation at the atomic level. In a model system, we have successively mapped the factor Xa binding site onto trypsin, showing that certain mutations influence both protein structure and inhibitor specificity. Our previous studies have shown that introduction of the 172SSFI175 sequence of factor Xa into rat or bovine trypsin results in the destabilisation of the intermediate helix with burial of Phe174 (the down conformation). Surface exposure of the latter residue (the up conformation) is critical for the correct formation of the aromatic box found in factor Xa-ligand complexes. In the present study, we investigate the influence of aromatic residues in position 174. Replacement with the bulky tryptophan (SSWI) shows reduced affinity for benzamidine-based inhibitors (1) and (4), whereas removal of the side-chain (alanine, SSAI) or exchange with a hydrophilic residue (arginine, SSRI) leads to a significant loss in affinity for all inhibitors studied. The variants could be crystallised in the presence of different inhibitors in multiple crystal forms. Structural characterisation of the variants revealed three different conformations of the intermediate helix and 175 loop in SSAI (down, up and super-up), as well as a complete disorder of this region in one crystal form of SSRI, suggesting that the compromised affinity of these variants is related to conformational flexibility. The influence of Glu217, peripheral to the ligand-binding site in factor Xa, was investigated. Introduction of Glu217 into trypsin variants containing the SSFI sequence exhibited enhanced affinity for the factor Xa ligands (2) and (3). The crystal structures of these variants also exhibited the down and super-up conformations, the latter of which could be converted to up upon soaking and binding of inhibitor (2). The improved affinity of the Glu217-containing variants appears to be due to a shift towards the up conformation. Thus, the reduction in affinity caused by conformational variability of the protein target can be partially or wholly offset by compensatory binding to the up conformation. The insights provided by these studies will be helpful in improving our understanding of ligand binding for the drug design process.

Figure 2.
Figure 2. Stereo view showing the alternative binding modes adopted by inhibitor (4) in the two structures (a) X(SSYI)bT.A4 and (b) X(SSYI)bT.B4. In a, the glycine spacer hydrogen bonds to Gly216; the tosyl group of the inhibitor occupies the S3/S4 site. In b, the glycine spacer hydrogen bonds to Gly219; the tosyl group points away from the enzyme, making contacts with a symmetry-related molecule in the crystal (not shown).

Figure 5.
Figure 5. a, Stereo view of the superposition of X(SSRI)bT.C1 (pink) and factor Xa (silver). For simplicity, the inhibitor (1) is not shown. Note the re-registration of residues Ser171-Ser178 and the formation of a hydrogen bond between Ser178 O and Asn233 Nd2 in the case of X(SSRI)bT.C1. b, Stereo diagram of the experimental electron density for X(SSRI)bT.B4; residues 169-175 (green) are disordered, and only partial density is present for Trp215 (violet). The ligand (4) is well defined, binding as seen in Figure 2b. Density for the cystine Cys168-Cys182 (orange) corresponds to the up conformation.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 335, 1325-1341) copyright 2004.

Secondary reference #1

Title

Zz made ez: influence of inhibitor configuration on enzyme selectivity.

Authors

D.Rauh, G.Klebe, J.Stürzebecher, M.T.Stubbs.

Ref.

J Mol Biol, 2003, 330, 761-770. [DOI no: 10.1016/S0022-2836(03)00617-X]

PubMed id

12850145

Figure 4.
Figure 4. Conformations of the cycloheptanone ring system viewed along the carbonyl axis. The isomers (E,E) (A, twisted) and (Z,Z) (B, chair) show a C[2] point symmetry with opposing twists of the ring, whereas isomer (E,Z) (C, asymmetric boat) lacks rotational symmetry. The carbonyl C1, C2 and C7 atoms are coloured silver.

Figure 5.
Figure 5. Binding modes of the three isomers transferred to the active site of factor Xa, together with solvent-accessible surfaces. (A) The cycloheptanone-ring system of the (E,Z)-isomer (light blue) clashes (yellow) with the side-chain of Tyr99 (red). (B) The (Z,Z)-configuration fits optimally to the binding site of factor Xa. (C) The (E,E) isomer makes no favourable interaction outside of the primary specificity pocket.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #2

Title

Trypsin mutants for structure-Based drug design: expression, Refolding and crystallisation.

Authors

D.Rauh, S.Reyda, G.Klebe, M.T.Stubbs.

Ref.

Biol Chem, 2002, 383, 1309-1314.

PubMed id

12437122

Secondary reference #3

Title

Reconstructing the binding site of factor xa in trypsin reveals ligand-Induced structural plasticity.

Authors

S.Reyda, C.Sohn, G.Klebe, K.Rall, D.Ullmann, H.D.Jakubke, M.T.Stubbs.

Ref.

J Mol Biol, 2003, 325, 963-977. [DOI no: 10.1016/S0022-2836(02)01337-2]

PubMed id

12527302

Figure 3.
Figure 3. (a) Experimental 2Fo 2 Fc electron density, contoured at a level of 1s, for co-crystals of (3) with X99rT. One half of the desired aromatic box is formed by the side-chains of Y99 and W215. (b) Soaking of the crystals with (1) reveals the inhibitor to bind in an extended conformation, with its chloronaphthyl group buried deep in the primary specificity pocket and the piperidinyl and piperidinyl rings in the position of the hydrophobic box of factor Xa. The side-chain of Y217 rotates from its position seen in (3) --X99rT so that its phenolic moiety approaches the hydro- phobic/aromatic moieties of the inhibitor.

Figure 5.
Figure 5. (a) Ribbon representation showing the orientation of the intermediate helix (blue) in wild-type rat trypsin; colour coding and orientation as in Figure 1. ( p ) Indicates the cystine C168-C182, which is in a right-handed helical conformation. Only side-chains of selected residues are shown for clarity. (b) In X(99/175/190)rT --(3) (yellow), the helix is tilted by ca 208, with unwinding of the final turn. Cystine C168-C182 ( p ) isomerises to an extended fully trans form, while F174 becomes buried in the body of the enzyme. (c) Stereo overlay of X(99/175/190)rT -- (3) and wild- type rat trypsin, showing the cavity formed by the disulphide and the side-chains of I176, W215, P225 and V227. The aromatic side-chain of F174 in X(99/175/190)rT -- (3) superimposes with that of trypsin Y172.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #4

Title

Ph-Dependent binding modes observed in trypsin crystals: lessons for structure-Based drug design.

Authors

M.T.Stubbs, S.Reyda, F.Dullweber, M.Möller, G.Klebe, D.Dorsch, W.W.Mederski, H.Wurziger.

Ref.

Chembiochem, 2002, 3, 246-249.

PubMed id

11921406

Secondary reference #5

Title

Structural and functional analyses of benzamidine-Based inhibitors in complex with trypsin: implications for the inhibition of factor xa, Tpa, And urokinase.

Authors

M.Renatus, W.Bode, R.Huber, J.Stürzebecher, M.T.Stubbs.

Ref.

J Med Chem, 1998, 41, 5445-5456. [DOI no: 10.1021/jm981068g]

PubMed id

9876114