Tài liệu Báo cáo khoa học: N-terminal extension of the yeast IA3 aspartic proteinase inhibitor

N-terminal extension of the yeast IA3 aspartic proteinase

inhibitor relaxes the strict intrinsic selectivity

Tim J. Winterburn1

, Lowri H. Phylip1

, Daniel Bur2

, David M. Wyatt1

, Colin Berry1 and John Kay1

1 School of Biosciences, Cardiff University, UK

2 Actelion Pharmaceuticals Ltd, Allschwil, Switzerland

Gene-encoded inhibitors of aspartic proteinases are

rather rare in nature. Thus, there is a need to under￾stand the mechanisms of action of the few that are

known, in order to exploit their therapeutic potential

[1]. We have described previously one such inhibitor:

the IA3 protein from Saccharomyces cerevisiae [1–4].

This remarkable polypeptide not only is a highly

potent inhibitor of its target enzyme, saccharopepsin,

but also appears to be completely specific for this sole

target proteinase [1,2]. Crystal structures solved for

complexes of IA3 with saccharopepsin revealed an

unprecedented mechanism of action [2,3]. IA3 from

S. cerevisiae consists of 68 residues but all of the inhib￾itory activity towards saccharopepsin resides within the

N-terminal half or segment of the polypeptide [2,3].

The free inhibitor is essentially unstructured [5,6] but,

upon contacting its target enzyme, residues 2–32

become ordered and adopt an alpha helical conforma￾tion occupying the active site cleft of the proteinase

[2,3]. This absolute selectivity for saccharopepsin was

shown to be conferred by a combination of the K18

and D22 residues in the S. cerevisiae IA3 sequence

Keywords

aspartic proteinase inhibition; IA3; inhibitor

engineering; Pichia aspartic proteinase;

specificity relaxation

Correspondence

J. Kay, School of Biosciences, Cardiff

University, Museum Avenue, Cardiff CF10

3US, UK

Fax: +44 029 20 87 41 16

Tel: +44 029 20 87 41 24

E-mail: [email protected]

(Received 30 March 2007, revised 23 May

2007, accepted 25 May 2007)

doi:10.1111/j.1742-4658.2007.05901.x

Yeast IA3 aspartic proteinase inhibitor operates through an unprecedented

mechanism and exhibits a remarkable specificity for one target enzyme, sac￾charopepsin. Even aspartic proteinases that are very closely similar to

saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not sus￾ceptible to significant inhibition. The Pichia proteinase was selected as the

target for initial attempts to engineer IA3 to re-design the specificity. The

IA3 polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii

differ considerably in sequence. Alterations made by deletion or exchange

of the residues in the C-terminal segment of these polypeptides had only

minor effects. By contrast, extension of each of these wild-type and chimaer￾ic polypeptides at its N-terminus by an MK(H)7MQ sequence generated

inhibitors that displayed subnanomolar potency towards the Pichia enzyme.

This gain-in-function was completely reversed upon removal of the exten￾sion sequence by exopeptidase trimming. Capture of the potentially posi￾tively charged aromatic histidine residues of the extension by remote,

negatively charged side-chains, which were identified in the Pichia enzyme

by modelling, may increase the local IA3 concentration and create an

anchor that enables the N-terminal segment residues to be harboured in clo￾ser proximity to the enzyme active site, thus promoting their interaction. In

saccharopepsin, some of the counterpart residues are different and, consis￾tent with this, the N-terminal extension of each IA3 polypeptide was with￾out major effect on the potency of interaction with saccharopepsin. In this

way, it is possible to convert IA3 polypeptides that display little affinity

for the Pichia enzyme into potent inhibitors of this proteinase and thus

broaden the target selectivity of this remarkable small protein.

Abbreviations

Nph, L-nitrophenylalanine; PpPr, vacuolar aspartic proteinase from Pichia pastoris; Z, L-norleucine.

FEBS Journal 274 (2007) 3685–3694 ª 2007 The Authors Journal compilation ª 2007 FEBS 3685