PDBsum entry 1qlq

Serine protease inhibitor

PDB id: 1qlq

Contents

Protein chain

58 a.a. *

Ligands

SO4 ×4

Waters ×98

* Residue conservation analysis

References listed in PDB file

Key reference

Title

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

Authors

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

Ref.

J Mol Biol, 2000, 295, 1237-1249. [DOI no: 10.1006/jmbi.1999.3445]

PubMed id

10653700

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.

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.

Secondary reference #1

Title

Structure of bovine pancreatic trypsin inhibitor at 125 k definition of carboxyl-Terminal residues gly57 and ala58.

Authors

S.Parkin, B.Rupp, H.Hope.

Ref.

Acta Crystallogr D Biol Crystallogr, 1996, 52, 18-29. [DOI no: 10.1107/S0907444995008675]

PubMed id

15299722

Figure 4.
Fig. 4. Electron densities contoured at i.0~ and 0.5a (coefficients Fobs) at the tw residues (a) Glu7 and (b) Arg53 for which refined disorder models were included.

Figure 7.
Fig. 7. Electron densities contoured at la, 3a and 5a (coefficients Fobs.) in regions with high refined thermal parameters at 125 K, the side chains of residues (a) Asp3, (b) Lysl5, (c) Arg39 and (d) Arg42.

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

Secondary reference #2

Title

Determination of a high-Quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures.

Authors

K.D.Berndt, P.Güntert, L.P.Orbons, K.Wüthrich.

Ref.

J Mol Biol, 1992, 227, 757-775. [DOI no: 10.1016/0022-2836(92)90222-6]

PubMed id

1383552

Figure 4.
Figure 4. Stereo view f the all-heavy-atom representations of the 3 crystal structures of PTI after superposition for minimal RMSD of the backbon atoms N, C'' and C' of residues 2 to 50. Thn line: form I (Deisenhofer & Steigemann, 1975); medium line: form II (Wlodawer t al., 1984): thick line: form III (Wlodawer et al., 19873). Same orientation of the olecule as shown in Fi. 2.`

Figure 6.
Figure 6. Location of NOE pper distance constraints measured for BPTI n solution that are violated by ore than @5 A in the X-ray crystal structur of BPTI form An all-heavy-atom representation of th X-ray crystal structure in he same orientation as shown in Fig. 2 is drawn with thin lines. NOE distance constraint violations are indicated with hick lines. The direction of these lines is that of a vector connecting the 2 hydrogen positions linked by the NOE, the ength is proportional to the extent of the violation. These NOES and the corresponding distances in the rystal structure re listed in Table 5.

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

Secondary reference #3

Title

Structure of bovine pancreatic trypsin inhibitor. Results of joint neutron and X-Ray refinement of crystal form ii.

Authors

A.Wlodawer, J.Walter, R.Huber, L.Sjölin.

Ref.

J Mol Biol, 1984, 180, 301-329. [DOI no: 10.1016/S0022-2836(84)80006-6]

PubMed id

6210373

Figure 6.
FIG. 6. Plots of the sums of neutron scttering densities for he hydrogen atoms of terminal methyl groups of Ala27, Ala16 and Thrll, expressed as functions of the orsion angles for these groups. Details of the caculations are given in the text.

Figure 11.
FIG. 11. Optimal superposition of C~ atoms in the of crystal form I (thin lines) and crystal form II (thick lines).

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

Secondary reference #4

Title

Crystallographic refinement of the structure of bovine pancreatic trypsin inhibitor at 1.5 a resolution

Authors

J.Deisenhofer, W.Steigemann.

Ref.

acta crystallogr ,sect b, 1975, 31, 238.