Tài liệu Báo cáo khoa học: An engineered disulfide bridge mimics the effect of calcium to protect

An engineered disulfide bridge mimics the effect of

calcium to protect neutral protease against local unfolding

Peter Du¨rrschmidt*, Johanna Mansfeld and Renate Ulbrich-Hofmann

Department of Biochemistry ⁄ Biotechnology, Martin-Luther University Halle-Wittenberg, Halle ⁄ Saale, Germany

The neutral protease from Bacillus stearothermophilus

belongs to a group of metalloendopeptidases that have

maximum activity at neutral pH. Some members of

this group are highly conserved in amino acid sequence

and tertiary structure and form the group of thermo￾lysin-like proteases (TLPs) with thermolysin as the best

characterized representative [1]. TLPs consist of 300–

319 amino acid residues and are organized into two

domains. They have one catalytic zinc ion, and

between two and four stabilizing calcium ions. The

X-ray structures of thermolysin [2] and the neutral

protease from B. cereus [3] have been resolved and

show great similarities. Because of the high degree of

sequence identity (85%) with thermolysin [4], a 3D

model of the neutral protease from B. stearothermophi￾lus (Fig. 1) was constructed, on the basis of the X-ray

structure of thermolysin, by homology modeling [5]

and has been successfully used in a number of

Keywords

autoproteolysis; disulfide; local unfolding;

neutral protease; stability

Correspondence

R. Ulbrich-Hofmann, Martin-Luther￾University Halle-Wittenberg, Department of

Biochemistry ⁄ Biotechnology, Institute of

Biotechnology, Kurt-Mothes-Strasse 3,

D-06120 Halle ⁄ Saale, Germany

Fax: +49 345 5527303

Tel: +49 345 5524864

E-mail: ulbrich-hofmann@biochemtech.

uni-halle.de

Enzymes

Neutral protease from Bacillus stearother￾mophilus (EC 3.4.24.28).

*Present address

IBFB Pharma GmbH, Deutscher Platz 5d,

D-04103 Leipzig, Germany

Note

A website is available: http://www.

biochemtech.uni-halle.de/biotech

(Received 9 December 2004, revised 26

January 2005, accepted 2 February 2005)

doi:10.1111/j.1742-4658.2005.04593.x

The extreme thermal stabilization achieved by the introduction of a disul￾fide bond (G8C ⁄ N60C) into the cysteine-free wild-type-like mutant (pWT)

of the neutral protease from Bacillus stearothermophilus [Mansfeld J, Vri￾end G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich￾Hofmann R & Eijsink VG (1997) J Biol Chem 272, 11152–11156] was

attributed to the fixation of the loop region 56–69. In this study, the role

of calcium ions in the guanidine hydrochloride (GdnHCl)-induced unfold￾ing and autoproteolysis kinetics of pWT and G8C ⁄ N60C was analyzed

by fluorescence spectroscopy, far-UV CD spectroscopy and SDS ⁄ PAGE.

First-order rate constants (kobs) were evaluated by chevron plots (ln kobs

vs. GdnHCl concentration). The kobs of unfolding showed a difference of

nearly six orders of magnitude (DDG# ¼ 33.5 kJÆmol)1 at 25
C) between

calcium saturation (at 100 mm CaCl2) and complete removal of calcium

ions (in the presence of 100 mm EDTA). Analysis of the protease variant

W55F indicated that calcium binding-site III, situated in the critical region

56–69, determines the stability at calcium ion concentrations between 5 and

50 mm. In the chevron plots the disulfide bridge in G8C ⁄ N60C shows a

similar effect compared with pWT as the addition of calcium ions, suggest￾ing that the introduced disulfide bridge fixes the region (near calcium

binding-site III) that is responsible for unfolding and subsequent autopro￾teolysis. Owing to the presence of the disulfide bridge, the DDG# is 13.2

kJÆmol)1 at 25
C and 5 mm CaCl2. Non-linear chevron plots reveal an

intermediate in unfolding probably caused by local unfolding of the loop

56–69. The occurrence of this intermediate is prevented by calcium concen￾trations of > 5 mm, or the introduction of the disulfide bridge

G8C ⁄ N60C.

Abbreviations

Abz-AGLA-Nba, 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide; DG#

, Gibbs free energy of activation; GdnHCl, guanidine hydrochloride;

kobs, observed rate constant; pWT, pseudo-wild type; TLP, thermolysin-like protease.

FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1523