Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation

Abstract

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.

Figure 1.

Ribbon diagram showing overall structure of the DPPIV homodimer, viewed perpendicular to the twofold dyad axis. Secondary structural elements that are involved in dimer formation are represented in red and in blue. The active site residues are shown as ball-and-stick representations. The α-helix comprising residues E205 and E206 is indicated in gold. The figure was made using the programs MOLSCRIPT (Kraulis 1991) and Raster3D (Merrit and Bacon 1997).

Figure 2.

(A) Surface representation of the β-propeller domain only, showing the propeller opening to the active site. The view was taken from the interface with the α/β-hydrolase domain and down the pseudo-eightfold axis. The four-strand antiparallel β-sheets of the eight blades are indicated (β1–β8). (B) Surface representation of whole DPPIV molecule, showing the side opening to the active site. Residues of DPPIV that make direct molecular interactions with tNPY are colored in both panels. Hydrophobic negatively charged and positively charged residues are shown in green, in red, and in blue, respectively. The figures were made with the program MOE (MOE, Chemical Computing Group).

Figure 3.

Stereo drawing of first six residues of Neuropeptide Y (magenta) and the underlying active site residues of DPPIV (pink) that make direct molecular interactions with the peptide. The peptide and selected DPPIV residues are shown as ball-and-stick representations. The peptide is not cleaved and trapped in a tetrahedral intermediate by which the carbonyl carbon is covalently linked to the active site S630. Hydrogen bonds are indicated as green dashed lines. The figure is made using the programs MOLSCRIPT (Kraulis 1991) and Raster3D (Merritt and Bacon 1997).

Figure 4.

Molecular surface representations showing the interaction of tNPY with DPPIV. Residues of the peptide are shown in ball-and-stick representations and DPPIV is shown as a solid surface. (A) Colors represent positive and negative electrostatic potential from blue (electropositive; white, neutral) to red (electronegative). (B) Colors represent hydrophobicity (green, polar; yellow, hydrophobic; white, exposed). The figures were made with the program MOE (MOE, Chemical Computing Group).

Figure 5.

Schematic representations showing tetrahedral intermediate formation. (A) 2Fo−Fc electron density map contoured at 1σ of the first six residues of tNPY and of the active site serine (S630). The peptide and the side chain of S630 are shown as ball-and-stick representations, and part of the DPPIV molecule is represented as a pink ribbon diagram. (B) Schematic representation showing the difference in conformation and hydrogen bond formation of active site residues S630 and H740 between the free form (green) of the enzyme and the tetrahedral intermediate (pink). The first three residues of the peptide are shown in gold as ball-and-stick representations. Hydrogen bonds are represented as green dotted lines, and measured distances are indicated in angstroms. Part of the DPPIV molecule around the active site is represented in pink as a ribbon diagram. The figures were made using the programs MOLSCRIPT (Kraulis 1991) and Raster3D (Merrit and Bacon 1997) and XtalView (McRee 1999).