PDBsum entry 1ld5

Hydrolase inhibitor

PDB id: 1ld5

Contents

Protein chain

58 a.a. *

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Nmr structures of two variants of bovine pancreatic trypsin inhibitor (bpti) reveal unexpected influence of mutations on protein structure and stability.

Authors

T.Cierpicki, J.Otlewski.

Ref.

J Mol Biol, 2002, 321, 647-658. [DOI no: 10.1016/S0022-2836(02)00620-4]

PubMed id

12206780

Abstract

Here we determined NMR solution structures of two mutants of bovine pancreatic trypsin inhibitor (BPTI) to reveal structural reasons of their decreased thermodynamic stability. A point mutation, A16V, in the solvent-exposed loop destabilizes the protein by 20 degrees C, in contrast to marginal destabilization observed for G, S, R, L or W mutants. In the second mutant introduction of eight alanine residues at proteinase-contacting sites (residues 11, 13, 17, 18, 19, 34, 37 and 39) provides a protein that denatures at a temperature about 30 degrees C higher than expected from additive behavior of individual mutations. In order to efficiently determine structures of these variants, we applied a procedure that allows us to share data between regions unaffected by mutation(s). NOAH/DYANA and CNS programs were used for a rapid assignment of NOESY cross-peaks, structure calculations and refinement. The solution structure of the A16V mutant reveals no conformational change within the molecule, but shows close contacts between V16, I18 and G36/G37. Thus, the observed 4.3kcal/mol decrease of stability results from a strained local conformation of these residues caused by introduction of a beta-branched Val side-chain. Contrary to the A16V mutation, introduction of eight alanine residues produces significant conformational changes, manifested in over a 9A shift of the Y35 side-chain. This structural rearrangement provides about 6kcal/mol non-additive stabilization energy, compared to the mutant in which G37 and R39 are not mutated to alanine residues.

Figure 2.
Figure 2. Comparison of calculated structures of BPTI_WT, BPTI_A16V and BPTI_8A. Upper pictures show the backbones of the ten lowest energy structures and lower pictures show their ribbon representation.

Figure 4.
Figure 4. Comparison of binding loop conformations (residues 11-18 and 34-40) of various BPTI mutants: BPTI_WT (PDB code 5pti), red; BPTI_A16V , green; BPTI_A16L (PDB code 1ejm), cyan; BPTI_G37A, blue. (a) Comparison of the effects of A16V and A16L mutations; (b) comparison of A16V and G37A mutations. For the reason of clarity only single conformers of the NMR structures are displayed.The Figure was prepared in MOLMOL/POV-Ray.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 321, 647-658) copyright 2002.

Secondary reference #1

Title

Substitutions at the p(1) position in bpti strongly affect the association energy with serine proteinases.

Authors

A.Grzesiak, R.Helland, A.O.Smalås, D.Krowarsch, M.Dadlez, J.Otlewski.

Ref.

J Mol Biol, 2000, 301, 205-217. [DOI no: 10.1006/jmbi.2000.3935]

PubMed id

10926503

Figure 1.
Figure 1. Inhibition curves of human a-plasmin by Ala16, Ala16 -> Gly and Ala16 -> Arg mutants of BPTI. Reactions were carried out in 100 mM Tris-Cl, 20 mM CaCl[2], 150 mM NaCl, 0.05 % Triton X-100 (pH 8.3) at 298 K. Data points were analyzed using the equations described in Materials and Methods.

Figure 7.
Figure 7. Stereo plot illustrating the competition between P'[1] and P'[3] residues for the same binding site. Red (inhibitor) and grey (trypsin) describes the bovine trypsin-BPTI P[1]-Lys complex while blue (inhibitor) and black (trypsin) describes the trypsin-BPTI P'[1]-Leu (molecule B) complex. The P[1], P'[1], P'[3] and P[4]' residues of the inhibitor are illustrated as ball and stick models. The Figures are generated using BOBSCRIPT [Kraulis 1991 and Esnouf 1997].

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