PDBsum entry 1qlq

Serine protease inhibitor

PDB id: 1qlq

Contents

Protein chain

58 a.a. *

Ligands

SO4 ×4

Waters ×98

* Residue conservation analysis

PDB id:

1qlq

Name:

Serine protease inhibitor

Title:

Bovine pancreatic trypsin inhibitor (bpti) mutant with altered binding loop sequence

Structure:

Pancreatic trypsin inhibitor. Chain: a. Synonym: bpti, aprotinin, trasylol, basic protease inhibitor. Engineered: yes. Mutation: yes

Source:

Bos taurus. Bovine. Organism_taxid: 9913. Organ: pancreas. Expressed in: escherichia coli. Expression_system_taxid: 469008. Expression_system_variant: t7

Resolution:

1.42Å R-factor: 0.110 R-free: 0.161

Authors:

H.Czapinska,S.Krzywda,G.M.Sheldrick,J.Otlewski,M.Jaskolski

Key ref:

H.Czapinska et al. (2000). High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence. J Mol Biol, 295, 1237-1249. PubMed id: 10653700 DOI: 10.1006/jmbi.1999.3445

Date:

10-Sep-99 Release date: 05-Oct-99

Protein chain

P00974  (BPT1_BOVIN) -  Pancreatic trypsin inhibitor from Bos taurus

Seq: 100 a.a.

Struc: 58 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

DOI no: 10.1006/jmbi.1999.3445

J Mol Biol 295:1237-1249 (2000)

PubMed id: 10653700

High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence.

H.Czapinska, J.Otlewski, S.Krzywda, G.M.Sheldrick, M.Jaskólski.

ABSTRACT

A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P(1) and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collected to 1.4 A at room temperature allowed anisotropic structure refinement characterized by R=0.1048. The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities. This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C(alpha) atoms of 0.7 A at Ala13). Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion. Three more SO(4)(2-) anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance. This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations. Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjacent intramolecular beta-hairpin loops, creates a four-stranded structure.

Selected figure(s)

Figure 5.
Figure 5. The 2mF[o] - DF[c] electron density showing the double-conformation Cys14-Cys38 disulfide bridge and the internal water molecule Wat1 with its tetrahedral hydrogen bonds.

Figure 7.
Figure 7. The 2-fold symmetric intermolecular b-sheet involving the 16-35 b-hairpins of two molecules. Note the role of water molecules sealing the gaps between the diverging strands on each end of the intermolecular b-sheet. For clarity, side-chains not involved in hydrogen bonding within this structure have been represented by their C^b atoms only.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 295, 1237-1249) copyright 2000.

Figures were selected by the author.