Structural Requirements for Substrate Specificity

In addition to information on the catalytic machinery, the first X-ray structures of PTP1B demonstrated a common binding mode of pTyr substrates: (1) binding of the phosphate moiety of pTyr to the P-loop (residues 214222) and Arg221, (2) hydrophobic interactions of the phenyl ring of pTyr with the active site pocket and (3) hydrogen bonds between the main chain nitrogens of the pTyr substrate (pTyr and residue +1) and the side chain of Asp48 of PTP1B, and (4) a hydrogen bond between the main chain carbonyl of residue -2 and the main chain nitrogen of residue 47 (Jia et al. 1995; Sarmiento et al. 1998). Specificity is provided by side chain interactions of the peptide and PTP1B (and other PTPs) (Yang et al. 2000), which has served as inspiration in early approaches to structure-based design of PTP inhibitors (see below).

A number of enzyme kinetic analyses with synthetic substrates and pep-tide-based inhibitors have provided insight into the fine specificity of PTPs (e.g., Cho et al. 1993; Ruzzene et al. 1993; Zhang et al. 1993; Zhang et al. 1994a; Desmarais et al. 1998; Desmarais et al. 1999). In an early and elegant study, Dixon and coworkers found that a catalytically inactive [35S]-labeled PTP1BC215S mutant, i.e., the first 'trapping mutant' (see below, Sect. 5), bound to activated, tyrosine phosphorylated epidermal growth factor receptor (EGF-R) with high affinity (Kd=100 nM) (Milarski et al. 1993). Synthetic tyrosine phosphorylated peptides corresponding to the four major au-tophosphorylation sites in the EGF-R were tested for their ability to displace PTP1BC215S from the full-length EGF-R. Although PTP1B was displaced by all four peptides, the peptides containing pTyr1148 or pTyr992 were the most potent. Peptides and peptide analogs corresponding to the EGF-R sequence around residue 992 (i.e., D-A-D-E-pY-L-I-P-Q-Q-G with pY=pTyr) have subsequently served as extremely useful tools in enzyme kinetic and structural analyses of PTP substrate specificity, and in inhibitor design, as will be discussed in the following. In a detailed analysis of the size and pTyr position ing requirements, it was found that efficient binding and catalysis was provided by Ac-D-A-D-E-pY-L-NH2 (Zhang et al. 1994a). A structural explanation for efficient binding of substrates with acidic residues immediately N-terminal to the pTyr residue was provided by the first X-ray structures of PTP1B in complex with this EGF-R-derived peptide, demonstrating specific interactions between the acidic residues and Arg47 of PTP1B (Jia et al. 1995; Sarmiento et al. 1998). Additional support for this notion was obtained by molecular modeling of D-A-D-E-X-L-based inhibitors in CD45 and LAR (Glover and Tracey 2000).

Using 'inverse alanine scanning,' Zhang and coworkers further assessed the substrate specificity of PTP1B (Vetter et al. 2000). Separate and sequential replacement of each alanine residue in the parent peptide, Ac-AAAApYAAAA-NH2, with the 19 other natural amino acids, identified Ac-ELEFpYMDYE-NH2 as a highly potent PTP1B substrate. As found previously, a strong preference was observed for acidic residues. Additionally, it was noted that PTP1B could also accommodate hydrophobic and aromatic residues at the -1 position. The structural basis for this plasticity was revealed by X-ray crystallographic studies showing that accommodation of both acidic and hydrophobic residues in the -1 position was conferred by Arg47, which can adopt different conformations, thereby generating two sets of distinct binding modes (Sarmiento et al. 2000).

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