Visualizing Active-Site Dynamics in Single Crystals of HePTP: Opening of the WPD Loop Involves Coordinated Movement of the E Loop

Abstract

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding/catalysis is comparatively unexplored. Hematopoietic tyrosine phosphatase (HePTP) is a member of the kinase interaction motif (KIM)-PTP family. Compared to the other PTPs, the KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loop in the transition between the closed and open states of HePTP, we identified a novel crystal form of HePTP that allowed the closed-to-open state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an ‘atypically open’ conformation and, importantly, that ligands can be exchanged at the active site, critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel, secondary site and function to coordinate the PTP, WPD and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for coordinated dynamics of the WPD and E loops in the catalytic cycle which, as we show, are relevant to multiple PTP families.

Frequency Response of a Protein to Local Conformational Perturbations

“…R-loop fluctuations may be given as a representative example to clarify this issue. Although difference in R-loop conformations between WPD open and WPD closed crystal structures is not significant, importance of R-loop mobility was recognized previously for WPD loop transitions [46], [79]. In our analysis, most of the R-loop backbone and side-chain dihedral angles was in the second and third cluster, i.e.…”

“…TMD force on each atom in the subset is computed by the gradient of the following potential:where RMSD( t ) is the RMSD between the current and target coordinates, and RMSD * ( t ) is a positive scalar linearly decreasing from the value of the initial RMSD between the first and target structures to zero. In the current study, TMD potential was initially applied on WPD loop atoms only, but side-chain of Glu115 on R-loop was seen to hinder the closure of WPD loop [46], [79], so TMD potential was extended to include R-loop atoms also. Smallest spring constant k rendering periodic WPD loop transitions [91] was found to be 3000 kcal·mol −1 ·Å −2 by trial and error.…”

SHP Family Protein Tyrosine Phosphatases Adopt Canonical Active-Site Conformations in the Apo and Phosphate-Bound States

“…In the WPD loop open state found in the SHP1 APO structure, the conserved R459 from the PTP signature motif is in an extended conformation and makes a salt bridge with the highly conserved E355 in the E loop (β5-β6 loop), a contact previously described as important for maintaining the E loop’s stability [14]. The aliphatic portions of R459 also make van der Waals contacts with the conserved WPD loop tryptophan, W417 (Figure 4A).…”

“…The aliphatic portions of R459 also make van der Waals contacts with the conserved WPD loop tryptophan, W417 (Figure 4A). These interactions are well conserved among other PTPs [12, 14, 33, 34]. …”

“…Structural analysis of phosphatase domain structures in both the WPD loop open and closed states have yielded descriptions of WPD loop conformational changes in PTP1B [12, 13], HePTP [14], and bacterial phosphatase YopH [15]. However, for the SHP family of phosphatases, there is currently not a structural description of the WPD loop transition from the ‘open’ to ‘closed’ state.…”