Distal mutation V486M disrupts the catalytic activity of DPP4 by affecting the flap of the propeller domain

Abstract

Dipeptidyl peptidase-4 (DPP4) plays a crucial role in regulating the bioactivity of glucagon-like peptide-1 (GLP-1) that enhances insulin secretion and pancreatic β-cell proliferation, making it a therapeutic target for type 2 diabetes. Although the crystal structure of DPP4 has been determined, its structure-function mechanism is largely unknown. Here, we examined the biochemical properties of sporadic human DPP4 mutations distal from its catalytic site, among which V486M ablates DPP4 dimerization and causes loss of enzymatic activity. Unbiased molecular dynamics simulations revealed that the distal V486M mutation induces a local conformational collapse in a β-propeller loop (residues 234–260, defined as the flap) and disrupts the dimerization of DPP4. The “open/closed” conformational transitions of the flap whereby capping the active site, are involved in the enzymatic activity of DPP4. Further site-directed mutagenesis guided by theoretical predictions verified the importance of the conformational dynamics of the flap for the enzymatic activity of DPP4. Therefore, the current studies that combined theoretical modeling and experimental identification, provide important insights into the biological function of DPP4 and allow for the evaluation of directed DPP4 genetic mutations before initiating clinical applications and drug development.

Biochemical characterization of a Mycobacterium tuberculosis prolyl dipeptidyl peptidase that displays a remarkable thermostability

“…MtDPP showed mild differences between the observed KM constants, whereas the turnover kcat values depicted significant disparities (p-value < 0.001; Table 1). In comparable trend to hDPP4 kinetics (8,23,27,28), MtDPP exhibits higher turnover rate kcat constant for the AMC surrogate in comparison to the pNA conjugated substrate (Table 1). Moreover, the three different h-Gly-Pro derivatives display substrate inhibition patterns that would suggest the relevance of the P1´ leaving group during catalysis (Table 1).…”

“…Alike hDPP4 (28), MtDPP displays remarkable thermal stability characterised by a melting temperature (Tm) of 70.4 ± 0.3 °C in thermal shift assays (TSA) (Fig. 2A).…”

“…3A). Considering its homology to human DPPs, the existence of a "Velcro" motif enabling a filter mechanism that increases selectivity for smaller peptides and the possible different conformations of the flap propeller loop next to the active site may both be structurally influencing catalysis (28,(30)(31)(32).…”

Role of Dipeptidyl Peptidase-4 (DPP4) on COVID-19 Physiopathology

“…DPP4 is a type II transmembrane glycoprotein [ 6 ] of 766 amino acids [ 7 , 8 ] and 110 kDa of molecular weight [ 9 ]. It is a serine exopeptidase belonging to the clan SC, family S9, and specifically to the S9B protein subfamily, based on the hierarchical and structural criteria of the MEROPS database [ 10 ].…”

“…The S9B subfamily is characterized by the motif Gly-Trp-Ser-Tyr-Gly-Gly-Tyr around the Ser630 located in the active site [ 13 ]. Other members belonging to this subfamily are fibroblast activation protein (FAP), resident cytoplasmic proteins (DPP8 and DPP9), and non-enzymatic members such as DPP6 and DPP10 [ 6 , 7 , 8 , 9 ]. They are also known as DPP4 activity and/or structure homologue (DASH) proteins.…”