Effect of protonation states of catalytically important residus and active site water molecules on PTP1B conformation

Abstract

Protein Tyrosine Phosphatases 1B (PTP1B) play a critical role in normal cell proliferation, differentiation, and metabolism, by removing the phosphate group from phosphotyrosine. Unliganded and liganded states of PTP1B are mostly associated with the open state of WPD loop (WPDopen) and the open state of WPD loop (WPDclosed) conformations in Protein Tyrosine Phosphatase (PTP) family, but in some cases WPD loop has been observed to adopt the closed conformation in the unliganded structures, and open conformation in the liganded structures. Position of the water molecules in the active site are suggested to help stabilize the closed conformation in PTP1B, and prevent the loop closure and maintain an atypical WPD loop conformation in STEP, which is another member of PTP family. Another controversial issue that may be related with the active site conformation and dynamics is the unusual protonation states of the active site residues Asp181 and Cys215: Asp181 is suggested to be protonated and Cys215 sidechain is suggested to be a thiolate. In this thesis, molecular dynamic (MD) simulations were used to study the effects of protonation states of Asp181 and Cys215, and the active site waters on the WPD loop conformations. MD simulations were performed on the unliganded PTP1B in WPDopen and WPDclosed, and the liganded PTP1B in WPDclosed conformations. In the WPDopen unliganded state, protonation state of Asp181 did not have a significant effect on the conformation or dynamics of the catalytically important loop regions in the vicinity of Asp181. The WPDclosed crystal structure conformation was maintained only in the MD simulations with a protonated Asp181 and with the initial positioning of crystal structure waters. With the active site waters missing, WPD loop moves from the closed to an intermediate conformation. Crystal structure conformations of the Michaelis complex were maintained only in the MD simulations with the protonated Asp181, protonated Cys215, and initialized with the single crystal structure water at the active site. When Cys215 was deprotonated, either the peptide or WPD loop moved out of the pocket. These results bring doubt on the controversial assumption that Cys215 should be in thiolate form in the Michaelis complex. In the absence of the active site water, Asp181 moves toward the position preoccupied by the water, and pTyr is slightly displaced. As a result, one may say that proper protonation states of active site residues and proper positioning of water molecules play important roles for the WPD loop activation/inactivation.