title: 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.
authors.affiliation: Department of Molecular Biosciences, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543-4000, USA. william.metzler@bms.com
authors.forename: William J
authors.initials: WJ
authors.lastname: Metzler
authors.forename: Joseph
authors.initials: J
authors.lastname: Yanchunas
authors.forename: Carolyn
authors.initials: C
authors.lastname: Weigelt
authors.forename: Kevin
authors.initials: K
authors.lastname: Kish
authors.forename: Herbert E
authors.initials: HE
authors.lastname: Klei
authors.forename: Dianlin
authors.initials: D
authors.lastname: Xie
authors.forename: Yaqun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Martin
authors.initials: M
authors.lastname: Corbett
authors.forename: James K
authors.initials: JK
authors.lastname: Tamura
authors.forename: Bin
authors.initials: B
authors.lastname: He
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Hamann
authors.forename: Mark S
authors.initials: MS
authors.lastname: Kirby
authors.forename: Jovita
authors.initials: J
authors.lastname: Marcinkeviciene
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004151:Dipeptides / Q000737:chemistry* / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry* / Q000378:metabolism; D054873:Dipeptidyl-Peptidase IV Inhibitors; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D020836:Protein Structure, Quaternary
chemical_list: D004151:Dipeptides; D054873:Dipeptidyl-Peptidase IV Inhibitors; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; C502994:saxagliptin; C042807:DPP4 protein, human; D018819:Dipeptidyl Peptidase 4; D000218:Adamantane