Ala⁶⁵⁷ and Conserved Active Site Residues Promote Fibroblast Activation Protein Endopeptidase Activity via Distinct Mechanisms of Transition State Stabilization

Abstract

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.

Fibroblast activation protein

“…13 Based on its structure, mutations of FAP with compromised protease activities have been engineered, including FAPS624A, which lacks dipeptidyl peptidase and endopeptidase activity, as well as FAPA657S, which retains its dipeptidyl peptidase activity but lacks endopeptidase activity. 14,15 These mutant forms of FAP provide important tools for defining the in vivo relevance of the distinct activities and physiologic substrates of FAP.…”

Pericyte-targeting prodrug overcomes tumor resistance to vascular disrupting agents

“…Previous researches have demonstrated that the conserved active site residues Arg123, Glu203, Glu204, Tyr656, and Asn704 are essential for the endopeptidase activity of FAPα (Protein Data Bank 1Z68) (15,19,20). We therefore constructed 5 mutant FAPα plasmids (R123A, E203A, E204A, Y656F, and N704A) to investigate whether Z-GP-DAVLBH can be specifically cleaved by FAPα.…”

Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice

“…Also, the widespread expression of many of these enzymes is likely to limit their potential as therapeutic targets. In contrast, FAP (also called FAPα or seprase) has recently gained attention as a potential target, due to its tightly regulated expression in the tumor stroma and structurally defined proteolytic activity (7)(8)(9)(10)(11); however, its function in tumors is largely unknown.…”

“…25,26). In vitro studies have shown that FAP has both dipeptidyl peptidase (19,24) and endopeptidase activity (8,10,27), including a collagenolytic activity capable of degrading gelatin (28,29) and type I collagen (27,30), but its in vivo substrate(s) is yet to be defined. Based on the highly regulated expression and restricted distribution of FAP, it has been suggested that FAP inhibition may be useful in cancer therapeutics.…”