PTP catalytic mechanism the general acid the general base

WPD loop D to A mutant

DA (trapping) PTPs WT (active) PTPs

Background PTP1B Knock Out Mice PTP1B -/- mice display enhanced sensitivity to insulin PTP1B -/- mice are resistant to development of obesity and Type 2 diabetes (Elchebly et al. Science (1999) 83, 1544-1548) (Klaman et al. Mol Cell Biol (2000) 20, 5479-89)

Inhibition of the Insulin Receptor PTP may Enhance Insulin Signaling Prolonged or Enhanced Signal Transient Signal

Structure of the PTP1B-IR peptide complex G1166 R1164 Phe182 Arg24 Arg254 pY1163 pY1162 Asp48 D1161 Tyr46 To understand What makes the bis and tris such good PTP1B substrate we solved the structure of PTP1B in complex with the IR peptides. Now, this is a surface representation of PTP1B in complex with the bisphospho peptide. The recognition of the substrate phosphotyrosine 1162 is similar to that for the EGFR-peptide where the pTYr extend into the catalytic site. The binding of the substrate pTyr induces closure of the WPD loop with Phen182 and Tyr46 sandwiching the tyrosine ring of pTYr 1162. As in the PTP1B-EGFR complex the depth of the catalytic cleft is defined by Tyr 46 and Nterminal acidic residues interact with PTP1B and orient the peptide Thus, Asp 1161 of the IR peptide interacts with and Arg47. The length of pTyr1163 to make the salt bridge interactions with Arg24 and 254 The amide groups of pTYr 1162 and 1163 interacts with the carboxylate side chain of Asp 48. Now What makes the bis and tris phophopeptides such good substrates is the salt bridge interactions Arg24 and 254 with the phosphoryl group of 1163 – In constratrs to the active site phosphotyrosine there is little interactions the phenyl ring From structural alignment studies PTP1B appears to be unique in having a wide cleft that allows access of peptides to this second binding site. In PTPalpha and LAR – which also have been implicated in IR signalling – this cleft is blocked by bulky residues and these PTPs would not recognizes a tandem pTYr motif with high affinity. Finally, at the very C-terminal Arg1164 forms a pi-cation interaction with the phenyl ring of Phe182 of the WPD loop which requires the loop to be closed ans Gly1166 is also defined in the structure. Arg47

IRTK Activation segment: DIpY1158ETDpY1162pY1163RKGG S.R. Hubbard, (1997) EMBO J., 16, 5573-5581

Kinetic Constants for Dephosphorylation of IR Activation Loop Peptide by PTP1B IR-peptide Km [mM] pY-1158 > 100 pY-1162 pY-1163 pY-1162,1163 14 ± 1.1 pY-1158, 1162,1163 7.9 ± 1.3 RDIpY1158ETDpY1162pY1163RKGGKGLL

PTP1B and insulin signaling Insulin Receptor PTK Signaling Response PTP1B

Suppression of PTP1B expression by RNAi may enhance tyrosine phosphorylation of IR 972 1158 PTP 1162 1163 Prolonged or Enhanced Signal Transient Signal