Tài liệu Báo cáo khoa học: Stefin A displaces the occluding loop of cathepsin B only by as much as

Stefin A displaces the occluding loop of cathepsin B only

by as much as required to bind to the active site cleft

Miha Renko, Ursˇka Pozˇgan, Dusˇana Majera and Dusˇan Turk

Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia

Introduction

Cathepsin B (EC 3.4.22.1), a lysosomal, papain-like

cysteine protease, is one of the most extensive studied

human cathepsins [1]. This enzyme is abundantly

expressed in a variety of tissues where it takes part in

protein degradation and processing. It is involved in a

number of physiological and pathological processes,

such as intracellular protein degradation, the immune

response, prohormone processing, cancer and arthritis

[2–9]. Its proteolytic activity is regulated by stefins and

cystatins, which are endogenous inhibitors of cysteine

cathepsins [10]. Cathepsin B differs from other cathep￾sins by its dual role, exhibiting exo- as well as endo￾peptidase activity. The crystal structure of this human

enzyme [11] has revealed that an 20 residues long

insertion, termed the ‘occluding loop’, occupies the

part of the active site cleft on the primed side and

blocks access to the active site cleft beyond the S2¢

substrate binding site [11,12]. The occluding loop is

Keywords

cathepsin B; complex; conformational

flexibility; crystal structure; occluding loop;

stefin A

Correspondence

D. Turk, Department of Biochemistry and

Molecular and Structural Biology, Jozef

Stefan Institute, Jamova 39, SI-1000

Ljubljana, Slovenia

Fax: +386 1 477 3984

Tel: +386 1 477 3215

E-mail: [email protected]

Database

The coordinates and structure factors are

available in the Protein Data Bank database

under accession number 3K9M

(Received 14 June 2010, revised 11 August

2010, accepted 16 August 2010)

doi:10.1111/j.1742-4658.2010.07824.x

Cathepsin B (EC 3.4.22.1) is one of the most versatile human cysteine cath￾epsins. It is important for intracellular protein degradation under normal

conditions and is involved in a number of pathological processes. The

occluding loop makes cathepsin B unique among cysteine cathepsins. This

20 residue long insertion imbedded into the papain-like protease scaffold

restricts access to the active site cleft and endows cathepsin B with its

carboxydipeptidase activity. Nevertheless, the enzyme also exhibits endo￾peptidase activity and is inhibited by stefins and cystatins. To clarify the

structural properties of the occluding loop upon the binding of stefins, we

determined the crystal structure of the complex between wild-type human

stefin A and wild-type human cathepsin B at 2.6 A˚ resolution. The papain￾like part of cathepsin B structure remains unmodified, whereas the occlud￾ing loop residues are displaced. The part enclosed by the disulfide bridge

containing histidines 110 and 111 (i.e. the ‘lasso’ part) is rotated by 45

away from its original position. A comparison of the structure of the unli￾ganded cathepsin B with the structure of the proenzyme, its complexes with

chagasin and stefin A shows that the magnitude of the shift of the occlud￾ing loop is related to the size of the binding region. It is smallest in the

procathepsin structures and increases in the series of complexes with stefin

A and chagasin, although it has no impact on the binding constant. Hence,

cathepsin B can dock inhibitors and certain substrates regardless of the size

of the binding region.

Structured digital abstract

l MINT-7990451: Stefin-A (uniprotkb:P01040) and Cathepsin B (uniprotkb:P07858) bind

(MI:0407) by x-ray crystallography (MI:0114)

Abbreviation

PDB, Protein Data Bank.

4338 FEBS Journal 277 (2010) 4338–4345 ª 2010 The Authors Journal compilation ª 2010 FEBS