The structure of apo protein-tyrosine phosphatase 1B C215S mutant: More than just an S -> O change

Citation
G. Scapin et al., The structure of apo protein-tyrosine phosphatase 1B C215S mutant: More than just an S -> O change, PROTEIN SCI, 10(8), 2001, pp. 1596-1605
Citations number
53
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
10
Issue
8
Year of publication
2001
Pages
1596 - 1605
Database
ISI
SICI code
0961-8368(200108)10:8<1596:TSOAPP>2.0.ZU;2-A
Abstract
Protein-tyrosine phosphatases catalyze the hydrolysis of phosphate monoeste rs via a two-step mechanism involving a covalent phospho-enzyme intermediat e. Biochemical and site-directed mutagenesis experiments show that the inva riant Cys residue present in the PTPase signature motif (H/V)CX5R(S/T) (i.e ., C215 in PTP1B) is absolutely required for activity. Mutation of the inva riant Cys to Set results in a catalytically inactive enzyme, which still is capable of binding substrates and inhibitors. Although it often is assumed that substrate-trapping mutants such as the C215S retain, in solution, the structural and binding propel ties of wild-type PTPases, significant diffe rences have been found in the few studies that have addressed this issue, s uggesting that the mutation may lead to structural/conformational alteratio ns in or near the PTP1B binding site. Several crystal structures of ape-WT PTP1B, and of WT- and C215S-mutant PTP1B in complex with different ligands are available, but no structure of the apo-PTP1B C215S has ever been report ed. In all previously reported structures, residues of the PTPase signature motif have an identical conformation, while residues of the WPD loop (a su rface loop which includes the catalytic Asp) assume a different conformatio n in the presence or absence of ligand. These observations led to the hypot hesis that the different spectroscopic and thermodynamic properties of the mutant protein may be the result of a different conformation for the WPD lo op. We report here the structure of the apo-PTP1B C215S mutant, which revea ls that, while the WPD loop is in the open conformation observed in the apo WT enzyme crystal structure, the residues of the PTPases signature motif a re in a dramatically different conformation. These results provide a struct ural basis for the differences in spectroscopic properties and thermodynami c parameters in inhibitor binding observed for the wild-type and mutant enz ymes.