Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DP8).
Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8(D772), DP8(Y315), DP8(H434) and DP8(D435) and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8(D772A/E/S/V) affected catalysis but did not confer endopeptidase activity. Mutations of DP8(H434F), DP8(D435F) and DP8(Y315F) reduced catalytic activity. Furthermore, mutations to DP8(D772A/E/S/V), DP8(H434F), DP8(D435F) and DP8(Y315F) affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8(D772), DP8(H434) and DP8(D435) were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.
Pitman
Melissa R
MR
School of Biological Sciences, Flinders University, Adelaide, Australia.
Menz
R Ian
RI
Abbott
Catherine A
CA
D055162:Biocatalysis; D020134:Catalytic Domain; D030562:Databases, Protein; D019281:Dimerization; D004150:Dipeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry; D010450:Endopeptidases; D018093:Gelatinases / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity; D000099167:Fibroblast Activation Protein Alpha
D004150:Dipeptidases; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases; D018093:Gelatinases; D008565:Membrane Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha; C471241:DPP9 protein, human; C042807:DPP4 protein, human; C487997:DPP8 protein, human