Involvement of DPP-IV catalytic residues in enzyme-saxagliptin complex formation

Abstract

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an approximately 1000-fold reduction in affinity relative to DPP-IV WT, while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at approximately 14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.

Figure 1.

Chemical structures of (A) saxagliptin and (B) BMS-538305.

Figure 2.

SDS-PAGE of DPP-IV^WT (lane 1), DPP-IV^H740Q (lane 2), DPP-IV^S630A (lane 3), DPP-IV^Y547F (lane 4), and DPP-IV^Y547Q (lane 5). The 4%–12% NuPAGE gel was run under nonreducing conditions. Each lane contains 5 μg of protein.

Figure 3.

Illustrations of X-ray co-crystal structure of saxagliptin (magenta) complexed with DPP-IV. (A) Protein colored from blue (N terminus) to red (C terminus). Catalytic residues (Ser630, His740, and Asp708) are shown in gray. All atoms are shown for Trp629, Ser630, and Tyr631 (green but for Ser630) to outline the oxyanion hole. Other active-site residues are colored per the ribbons trace. Selected hydrogen-bonded water molecules are shown as red spheres. (B) Same as above in stereo and zoomed in on BMS-477118. The electron density (2F _o − F _c, 1σ, cyan) is consistent with covalent attachment to Ser630. Figure created with PyMOL (DeLano Scientific).

Figure 4.

Binding of saxagliptin to (A) DPP-IV^WT and (B) DPP-IV^S630A by ITC.

Figure 5.

Effect of saxagliptin (squares) and BMS-538305 (circles) on the melting temperature of DPP-IV^WT (filled symbols) and DPP-IV^S630A (open symbols).

Figure 6.

Expansion of the downfield ¹H NMR spectra of wild type and the four DPP-IV mutant proteins in the absence of any inhibitor (A) and in the presence of 1.2-molar excess BMS-538305 (B) or saxagliptin (C).