Protein Tyrosine Phosphatases (PTPs) play an important role in regulating protein tyrosine phosphorylation levels. NMR data has demonstrated a correlation between the general acid loop (WPD-loop) dynamics and catalytic function. However, what factors control loop dynamics and structure are unclear. To examine these factors, we have made loop-exchanged chimeras of two PTPs that have superimposable active sites, YopH and PTP1B. YopH (Yersinia pestis) and PTP1B (Homo sapiens) are the best characterized PTPs. YopH is a virulence factor for the “black plague” and is the fastest known PTP. PTP1B is essential for regulating the insulin signaling pathway via dephosphorylating the insulin receptor. Both PTPs are chemically and mechanistically indistinguishable, though PTP1B has proven to be more tolerant to mutations in the WPD-loop. Chimeras with the YopH loop transposed into PTP1B have been made and characterized kinetically and structurally. The structural integrity and catalytic rates are affected by the length of the transposed regions. One chimera exhibits a steady state rate resembling native PTP1B, while a closely related chimera displays slower enzymatic activity. Both chimeras showed conventional bell-shaped pH-rate profiles, similar to wild-type PTPs, indicating fully functional general acid catalysis by the conserved Asp 181 in the WPD-loop and nucleophilic Cys. The kinetic isotope effect results for both chimeras with the substrate pNPP suggest full restoration of general acid catalysis. Pre-steady state experiments show burst kinetics for one chimera, as for the wild-type, while the other does not, raising questions about a possible alteration of the rate determining step in the mechanism. X-ray crystal structures for both chimeras showed superimposable WPD loop open and closed conformations with native PTP1B, however, an unconventional conformation for the general acid Asp181 was captured, a result of different local interactions with a magnesium ion used in the crystallization conditions.

Insights from PTP1B/YopH chimeras into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases

2019 Fall National Meeting

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