Investigations into the effect of protein motions and small molecule inhibitors on the PTP YopH

Catalysis rates of protein tyrosine phosphatases (PTPs) vary by nearly four orders of magnitude despite structurally superimposable active sites and the same catalytic mechanism. The faster PTPs, including YopH, the fastest known, have a flexible WPD-loop containing the catalytic general acid Asp356 that closes over the active site for catalysis. Faster dynamics of this flexible loop are correlated to the faster YopH rate compared to other PTPs. Mutation of the tryptophan 354 residue in the WPD-loop to phenylalanine, tyrosine, or histidine, locks the loop in a quasi-open position. These mutations cause a \u3e 100-fold decrease in rate and loss of general acid catalysis. We report the highest resolution structure of native YopH to date (1.05 Â), and structures of the W354Y and W354H mutants between 1.1-1.3 Â. Crystals of the mutants grown with vanadate show a divanadate glycerol ester in the active site, formed after glycerol was added as a cryoprotectant. Kinetics data show glycerol has no synergistic effect on vanadate inhibition, suggesting the absence of glycerol-vanadate esters as inhibitory species in solution. The electrostatics of the active site and the disabled general acid in these mutants provides a unique environment for the formation of the unusual ester

Investigations into Factors Affecting the WPD-Loop in the Protein Tyrosine Phosphatases YopH and PTP1B

The research in this dissertation documents connections between the primary amino acid sequence of proteins, the dynamics of proteins, and their catalytic function. This research project studied two proteins called protein-tyrosine phosphatases (PTPs): the human enzyme PTP1B, and the bacterial enzyme YopH. PTP1B is a human enzyme that down regulates the insulin receptor on the outer cellular membrane, and causes the insulin receptor to be less responsive to insulin. A deeper knowledge of how PTP1B is different from other human PTPs might be useful in designing drugs to increase insulin sensitivity in diabetics. Yersinia Pestis is the bacteria that caused the Black Plague, and YopH is an essential for virulence factor that helps Yersinia Pestis to escape the human immune response. Using these proteins, the primary sequence of amino acids in a small but critical loop region was altered and the effect on the catalytic efficiency was measured. This research shows how some residues are key to the catalytic efficiency of PTPs while others could be changed with little to no effect on the catalytic efficiency. A deeper understanding of the difference between key residues and structural residues may allow future scientists to create designer enzymes and perhaps design pharmaceuticals that mediate enzyme activity by affecting their protein dynamics

Conservative tryptophan mutants of the protein tyrosine phosphatase YopH exhibit impaired WPD-loop function and crystallize with divanadate esters in their active sites.

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution

Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution