PDBsum entry 1k9b

Hydrolase inhibitor

PDB id: 1k9b

Contents

Protein chain

58 a.a. *

Waters ×17

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Crystal structure of the bifunctional soybean bowman-Birk inhibitor at 0.28-Nm resolution. Structural peculiarities in a folded protein conformation.

Authors

R.H.Voss, U.Ermler, L.O.Essen, G.Wenzl, Y.M.Kim, P.Flecker.

Ref.

Eur J Biochem, 1996, 242, 122-131.

PubMed id

8954162

Abstract

The Bowman-Birk inhibitor from soybean is a small protein that contains a binary arrangement of trypsin-reactive and chymotrypsin-reactive subdomains. In this report, the crystal structure of this anticarcinogenic protein has been determined to 0.28-nm resolution by molecular replacement from crystals grown at neutral pH. The crystal structure differs from a previously determined NMR structure [Werner, M. H. & Wemmer, D. E. (1992) Biochemistry 31, 999-1010] in the relative orientation of the two enzyme-insertion loops, in some details of the main chain trace, in the presence of favourable contacts in the trypsin-insertion loop, and in the orientation of several amino acid side chains. The proximity of Met27 and Gln48 in the X-ray structure contradicts the solution structure, in which these two side chains point away from each other. The significant effect of a Met27-->Ile replacement on the inhibitory activity of the chymotrypsin-reactive subdomain agrees with the X-ray structure. Exposed hydrophobic patches, the presence of charged amino acid residues, and the presence of water molecules in the protein interior are in contrast to standard proteins that comprise a hydrophobic core and exposed polar amino acids.

Secondary reference #1

Title

Crystal structure of cancer chemopreventive bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 a resolution. Structural basis of janus-Faced serine protease inhibitor specificity.

Authors

J.Koepke, U.Ermler, E.Warkentin, G.Wenzl, P.Flecker.

Ref.

J Mol Biol, 2000, 298, 477-491. [DOI no: 10.1006/jmbi.2000.3677]

PubMed id

10772864

Figure 1.
Figure 1. Schematic representation of active site of bovine trypsin. The catalytic triad Ser195, His57 and Asp102 of the enzyme are shown in the centre. The NH group atoms of Ser195 and Gly193 forming the oxyanion hole were omitted for clarity. The walls 189-195, 214-220 and 225-228 and the surface loops 185-188 and 221-225 of the S1 pocket are highlighted in blue and in green-blue. The surface loops 90-104 (magenta), 140-156 (red) and 171-178 (violet) surrounding the S1 pocket are also highlighted. Residues 15-19 of sBBI are shown in orange. The side-chains of Leu99, Trp215 and Tyr172 forming the S4 pocket and those of Tyr151 and Gln192 (deleted beyond C^b forming the S2' pocket are shown. The side-chain of P1LysI16 interacting with Asp189 is shown. Amino acid side-chains are numbered near the C^a atoms, but in Asp189, Tyr151, Tyr172 and Trp215 they are numbered at their tips.

Figure 6.
Figure 6. Buried surface area. The buried surface area was viewed along the crystallographic 2-fold axis with subdomain 1 on the left and subdomain 2 on the right. Red, polar residues; green, hydrophobic residues. The two trypsin molecules are indicated by yellow lines.

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

Secondary reference #2

Title

Chemical synthesis, Molecular cloning and expression of gene coding for a bowman-Birk-Type proteinase inhibitor.

Author

P.Flecker.

Ref.

Eur J Biochem, 1987, 166, 151-156.

PubMed id

3297696

Secondary reference #3

Title

Mutational analysis of disulfide bonds in the trypsin-Reactive subdomain of a bowman-Birk-Type inhibitor of trypsin and chymotrypsin--Cooperative versus autonomous refolding of subdomains.

Authors

S.Philipp, Y.M.Kim, I.Dürr, G.Wenzl, M.Vogt, P.Flecker.

Ref.

Eur J Biochem, 1998, 251, 854-862. [DOI no: 10.1046/j.1432-1327.1998.2510854.x]

PubMed id

9490061

Figure 1.
Fig. 1. Replacements in the disulfide­bond framework of rBBI. The scissile peptide bonds in the trypsin (Tr) and the chymotrypsin­reactive subdomain (Ch) are indicated by arrows. The three loops encompassed by S­S bonds are indicated in roman letters. The mutations in the rBBI molecule are indicated as follows : circles, C14A, C22A; squares, C8A, C12A; hexagons, C9A, C24A; pentagons, C14T, T15C; triangles, N18C, Q21C. The #­strand A and B span between Asp10­Thr15 and Gln21­Cys24, respectively. C14A, C22A, C8A, C12A and C14T, T15C variants contain at least one mutation in strand A. C9A, C24A and N18C, Q21C variants are characterized by at least one replacement in strand B.

Figure 6.
Fig. 6. Overall fold and subdomain interface of BBI. The schematic phys. Res. Commun. 220, 2462254. representation of the crystal structure of BBI (Voss et al., 1996) was Clarke, J., Henrick, K. & Fersht, A. R. (1995) Disulfide mutants of generated from the crystal structure using the program MOLSCRIPT barnase I: changes in stability and structure assessed by biophysical (Kraulis, 1991). The trypsin­reactive subdomain is a discontinuous methods and X­ray crystallography, J. Mol. Biol. 253, 4932504. region (Wetlaufer, 1973) comprising amino acids Cys92Cys24 and Cordes, M. H. J., Davidson, A. R. & Sauer, R. T. (1996) Sequence space, Val522Cys62 as shown on the bottom. The chymotrypsin­reactive sub­ folding and protein design, Curr. Opin. Struct. Biol. 6, 3210. domain on the top is a continuous region containing residues Ser252 Dai, Y. & Tang, J. G. (1996) Characteristic activity and conformational Cys51. The scissile peptide bonds in the two subdomains is indicated by studies of [A6­Ser, A11­Ser]­insulin, Biochim. Biophys. Acta 1296, arrows. The main­chain trace is indicated by light gray and the two 63268. enzyme­insertion loops by dark gray lines. The #­strands of the two Dyson, H. J. & Wright, P. E. (1993) Peptide conformation and protein subdomains are represented by broad gray arrows. The disulfide bonds folding, Curr. Opin. Struct. Biol 3, 60265. are represented by bold lines and the S­S bonds deleted in the present Fersht, A. R. (1997) Nucleation mechanisms in protein folding, Curr. report are marked by italic numbers. The trypsin­reactive subdomain Opin. Struct. Biol. 7, 329. contains the #­strands A, B and F and the chymotrypsin­reactive subdo­ Flecker, P. (1987) Chemical synthesis, molecular cloning and expression main the #­strands C, D and E. The side­chains of Asp10 (#­strand A), of gene coding for a Bowman­Birk­type proteinase inhibitor, Eur. J. Asp26 and Arg28 (hydrophilic face of #­strand C), Gln48 and Asp53 Biochem. 166, 1512156. (#­strand F) are shown. His33 and Lys37 were omitted for clarity. The Flecker, P. (1989) A new and general procedure for refolding mutant significance of close ion pairs between the #­strands A and C is reflected Bowman­Birk­type proteinase inhibitors on trypsin­Sepharose as a by the dramatic effects of the class­I mutations on the chymotrypsin­ matrix with complementary structure, FEBS Lett. 252, 1532157. reactive subdomain. The absence of close ion pairs between B and the Flecker, P. (1995) Template­directed protein folding into a metastable chymotrypsin­reactive subdomain is reflected by less dramatic effects state of increased activity, Eur. J. Biochem. 232, 5282535. on the chymotrypsin­reactive subdomain in the class­II variants (a,c,d in Goldberg, M. E. & Guillou, Y. (1994) Native disulfide bonds greatly Fig. 4). The H­bond between S#Met27­N#2Gln48 on the hydrophobic accelerate secondary structure formation in the folding of lysozyme, face C is represented by a dotted line. Atoms are defined as follows : C, Protein Sci. 3, 8832887. black; O, dark gray; N, light gray (small); S, light gray (large). Grosjean, H. & Fiers, W. (1982) Preferential codon usage in prokaryotic genes : the optimal codon­anticodon interaction energy and the selec­ tive codon usage in efficiently expressed protein genes, Gene (Amst.) chymotrypsin­reactive material in the C9A, C24A variant could 18, 1992209. reflect the water­mediated hydrogen bonds between Cys24 and Harrison, P. M. & Sternberg, M. J. (1996) The disulphide #­cross : from His33 (Voss et al., 1996). The present data cannot rigorously cystine geometry and clustering to classification of small disulphide­ rich protein folds, J. Mol. Biol. 264, 6032623.exclude a relevance of loosely bound water molecules that are Harry, J. B. & Steiner, R. F. (1969) Characterisation of the self associa­ not observed in the crystal structure. tion of a soybean proteinase inhibitor by membrane osmometry, Bio­ Taken together, the results of the present report point to the chemistry 8, 506025064. significance of the polar domain interface as a major refolding Hinck, A. P., Truckses, D. M. & Markley, J. L. (1996) Engineered determinant of BBI. The observed effects seem to result from a disulfide bonds in Staphylococcal Nuclease: effects on the stability combination of close ion pairs and hydrogen bonds between and conformation of the folded protein, Biochemistry 35, 103282 strands A and C. Direct mutations of ion pairs (especially in 10338. positions 28 and 33) are required for a more clear­cut distinction Hua, Q. X., Narhi, L., Jia, W. H., Arakawa, T., Rosenfeld, R., Hawkins, between these two possibilities. However, we expect that this N., Miller, J. A. & Weiss, M. A. (1996) Native and non­native struc­ ture in a protein­folding intermediate : spectroscopic studies of par­could be complicated by compensatory interactions with other

The above figures are reproduced from the cited reference with permission from the Federation of European Biochemical Societies

Secondary reference #4

Title

Template-Directed protein folding into a metastable state of increased activity.

Author

P.Flecker.

Ref.

Eur J Biochem, 1995, 232, 528-535.

PubMed id

7556203

Secondary reference #5

Title

A new and general procedure for refolding mutant bowman-Birk-Type proteinase inhibitors on trypsin-Sepharose as a matrix with complementary structure.

Author

P.Flecker.

Ref.

febs lett, 1989, 252, 153.