Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.

Protein-tyrosine phosphatases can exhibit stringent substrate specificity in vivo, although the molecular basis for this is not well understood. The three-dimensional structure of the catalytically inactive protein-tyrosine phosphate 1B (PTP1B)/C215S complexed with an optimal substrate, DADEpYL-NH 2 , reveals specific interactions between amino acid residues in the substrate and PTP1B. The goal of this work is to rigorously evaluate the functional significance of Tyr 46 , Arg 47 , Asp 48 , Phe 182 , and Gln 262 in substrate binding and catalysis, using site-directed mutagenesis. Combined with structural information, kinetic analysis of the wild type and mutant PTP1B using p-nitrophenyl phosphate and phosphotyrosine-containing peptides has yielded further insight into PTP1B residues, which recognize general features, as well as specific properties, in peptide substrates. In addition, the kinetic results suggest roles of these residues in E-P hydrolysis, which are not obvious from the structure of PTP1B/peptide complex. Thus, Tyr 46 and Asp 48 recognize common features of peptide substrates and are important for peptide substrate binding and/or E-P formation. Arg 47 acts as a determinant of substrate specificity and is responsible for the modest preference of PTP1B for acidic residues NH 2 -terminal to phosphotyrosine. Phe 182 and the invariant Gln 262 are not only important for substrate binding and/or E-P formation but also important for the E-P hydrolysis step.Protein-tyrosine phosphorylation is a universal mechanism employed for the regulation of cellular processes such as proliferation, differentiation, motility, cell-cell interactions, metabolism, gene transcription, and the immune response (1, 2). The propagation and termination of signaling events controlling these cellular processes are determined by the level of phosphorylated proteins in a cell. The phosphorylation level, in turn, is maintained in an exquisite balance by the reciprocal activities of protein-tyrosine kinases and phosphatases. Thus, in addition to the study of protein-tyrosine kinases, one can appreciate the need to further characterize the dephosphorylation reaction catalyzed by the protein-tyrosine phosphatases (PTPases). 1Much is known about the catalytic mechanism of the PTPases (3). However, the molecular basis for PTPase substrate specificity is not well understood and remains a major unresolved issue in the field. The PTPase family is presently composed of approximately 100 enzymes, which can be either transmembrane (receptor-like) or intracellular (cytoplasmic). Membership in this family of enzymes requires the presence of the PTPase signature motif, (H/V)CX5R(S/T), housed within the catalytic domain. Outside this shared catalytic domain are various targeting and localization domains, which may be utilized for controlling and restricting PTPase substrate specificity. There have been relatively few biochemical analyses of the mechanisms that govern PTPase substrate specificity, although recent genetic and biochemical evidence sugg...

Protein tyrosine phosphatases (PTPases) are involved in the control of tyrosine phosphorylation levels in the cell and are believed to be crucial for the regulation of a multitude of cellular functions. A detailed understanding of the role played by PTPases in various signaling pathways has not yet been achieved, and potent and selective PTPase inhibitors are essential in the quest to determine the functionality of individual PTPases. Using the DOCK methodology, we have carried out a structure-based, computer-assisted search of an available chemical database in order to identify low molecular weight, nonpeptidic PTP1B inhibitors. We have identified several organic molecules that not only possess inhibitory activity against PTP1B but which also display significant selectivity for PTP1B. This indicates that although structural features important for pTyr recognition are conserved among different PTPases, it is possible to generate selective inhibitors targeted primarily to the catalytic site. Kinetic analysis and molecular modeling experiments suggest that the PTP1B active site possesses significant plasticity such that substituted and extended aromatic systems can be accommodated. The newly identified molecules provide a molecular framework upon which therapeutically useful compounds can ultimately be based, and systematic optimization of these lead compounds is likely to further enhance their potency and selectivity.