In the case of RPTPa, where both D1 and D2 domains are active, the phosphatase activity of the D2 domain is crucial for RPTPa to elicit its biological response . These apparently contradictory findings suggest that the role of the D2 domain could vary substantially. In this study, the substrate specificity of the tandem PTP domains of DLAR and PTP99A were examined using tyrosine phosphorylated peptides. In the case of DLAR, an analysis of PTP domain-peptide interactions suggests that the D2 domain binds to substrate peptides with a higher affinity than the D1 domain. In PTP99A, however, the D2 domain binds the peptides with a much lower affinity, when compared to its D1 domain. Fluorescence spectroscopy experiments using small molecule probes highlight the differences in the phosphotyrosine binding pockets of the two domains of DLAR and PTP99A. Molecular dynamics simulations using models of DLAR and PTP99A explain the lack of catalytic activity in the DLAR and PTP99A D2 domains, while providing a rationale for their substrate interaction. Importantly, critical differences in the inter-atomic interaction network rationalize the differences in the catalytic activities seen for the DLAR and PTP99A PTP domains. These studies thus suggest that the silent D2 domains may have evolved to provide a balance between peptide-binding and peptide-dephosphorylation in bi-domain PTPs. Cosmids containing the genes encoding RPTPs DLAR and PTP99A were a kind gift from Prof. Kai Zinn . The PTP domains of PTP99A and DLAR were PCR amplified and ligated between the NheI and XhoI restriction sites of the bacterial expression vectors pET-15b and pET-22b. The active site mutants were obtained by using a single primer approach. An XbaI site was incorporated in the primers to aid screening of mutants. All clones were confirmed by sequencing . The details of the expression constructs are BU 4061T Proteasome inhibitor compiled in Table S1 and Figure 1a. The plasmids containing the recombinant PTP domains were transformed into E. coli BL21 cells . Cells were grown to an optical density of 0.6 at 37uC in Luria Broth and induced with 0.1 mM IPTG.