The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

Abstract

The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 A crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated beta-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed beta-propeller with open Velcro topology and the C-terminal alpha/beta-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the beta-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu-Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P(2)-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.

Figure 1

Alignment of the newly determined porcine DP IV sequence with those of human and mouse DP IV as well as with the related FAP-α and prolyl oligopeptidase. The comparison rationalizes the lack of dimerization of POP and correlates the lack of ADA binding in rodents with the glycosylation site Asn-279 (281).

Figure 2

Soluble DP IV forms a 222 symmetric assembly as a dimer of dimers. The view is along one two-fold axis. Potential glycosylation sites are indicated as gray spheres, and red spheres are the sites modified in our crystal structure. The transmembrane helices and their orientation to the membrane were modeled to illustrate how tetramerization of DP IV can mediate cell–cell contacts. The figures were prepared by using the programs MAIN (25), MOLSCRIPT (55), and RASTER3D (56).

Figure 3

Topology diagram illustrating the domain structure of DP IV. Blade IV of the propeller is involved in both the dimer contact (IV A–IV B: L235-P255, together with the highlighted C-terminal three secondary structure elements F713-C762) and the tetramerization of DP IV (IV A–IV C and IV B–IV C, not shown).

Figure 4

View along the two-fold axis on the tetramerization interface. Blades IV of each subunit align to form an eight-bladed antiparallel β-sheet. The highlighted Leu-294 and Val-341 are involved in ADA binding.

Figure 5

Substrate recognition by DP IV. (a) The peptidomimetic inhibitor p-Iodo-Phe-Pyr-CN is covalently bound to active site Ser-630. The accessible surface is indicated and cut open (dark green) for better visibility. (b) Schematic representation of the active site access in tricorn and DP IV.