Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS

Abstract

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and buriedness. We demonstrate that our HDX rate data obtained in solution adds value to predictions of dynamics derived from a pseudo-ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site vs. allosteric small-molecule inhibitors. These maps reveal distinct, dramatic, and widespread effects on protein dynamics relative to the apo form, including changes to dynamics in locations distal (>35 Å) from the respective ligand binding sites. These results help shed light on the allosteric nature of PTP1B and the surprisingly far-reaching consequences of inhibitor binding in this important protein. Overall, our work showcases the potential of HDX-MS for elucidating protein conformational dynamics and allosteric effects of small-molecule ligands, and highlights the potential of integrating HDX-MS alongside other complementary methods to guide the development of new therapeutics.

Figure 1:. Overview of PTP1B structure and key sites.

A. Schematic of full-length PTP1B primary structure, including catalytic domain and disordered C-terminus. Key structural sites in the catalytic domain are labeled. The 1-321 construct used throughout this study is indicated below the diagram. The last ~22 residues of this construct do not appear in crystal structures. B. Superposed structures of the PTP1B catalytic domain bound to the active-site inhibitor TCS401 (green, PDB ID 5K9W, closed state) and bound to the allosteric inhibitor BB3 (purple, PDB ID 1T49, open state). C. Close-up view of PTP1B active site showing the positions of catalytic loops surrounding TCS401.

Figure 2:. High-resolution local HDX-MS map for apo PTP1B.

A. Woods plot of HDX rates for 312 peptides of apo PTP1B showing %deuteration at 30 seconds of labeling for each peptide. B. Deconvoluted HDX rates at 30 s of labeling time mapped to a crystal structure of the PTP1B catalytic domain in the closed state (PDB ID 1SUG). Several key loops and the α4 helix (residues 187-200) and α7 helix (residues 285-298) are indicated.

Figure 3:. Apo PTP1B local HDX-MS reaction rates are only partially explained by a pseudo-ensemble of crystal structures.

A. PTP1B pseudo-ensemble derived from all non-PanDDA crystal structures from the PDB (n=199; see Methods). Only protein chain A shown; colored from N-to C-terminus (blue to red). B. Plot of peptide-level HDX-MS %deuteration (back-exchange corrected) at the 30 second time point vs. pseudo-ensemble Cα root-mean-square fluctuation (RMSF). Average RMSF values are shown as circles. Range of individual residue RMSF values within each peptide are shown as bars. C-E. The three peptide groups based on criteria of Cα RMSF and HDX from panel B, mapped to a crystal structure of the PTP1B catalytic domain in the closed state (PDB ID 1SUG). C. Peptides with low mean RMSF (< 0.5 Å) and low HDX (< 5%), in blue. D. Peptides with low mean RMSF (< 0.5 Å) and high HDX (> 70%), in magenta. E. Peptides with high mean RMSF (> 1.25 Å), in orange.

Figure 4:. Difference Woods plot of active-site and allosteric inhibitors relative to apo PTP1B.

Difference in %deuteration values at 30 seconds in each inhibitor-bound state minus the apo PTP1B state, plotted against amino acid sequence. Blue lines indicate a difference interval of ± 5%. A. Active-site inhibitor, TCS401. B. Allosteric inhibitor, BB3.

Figure 5:. Effects of active-site inhibitor (TCS401) and allosteric site inhibitor (BB3) on local HDX.

The structure of PTP1B in complex with (A) the active-site inhibitor TCS401 (PDB ID 5K9W) and (B) the allosteric inhibitor BB3 (PDB ID 1T49) color-mapped with the deconvoluted differential HDX values at 30 seconds of labeling time. Particular peptides of interest have been selected and their fractional deuterium build-up plots shown. See also Fig. S3.