A three-dimensional model of the neprilysin 2 active site based on the X-ray structure of neprilysin. Identification of residues involved in substrate hydrolysis and inhibitor binding of neprilysin 2.
Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of the in vitro enzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S'(2) subsite of NEP2 where Ser(133) and Leu(739) replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg(131) and Asn(567) in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S'(2) pocket, particularly the presence therein of Leu(739), account for a number of differences in inhibitor binding between NEP and NEP2.
Unité de Neurobiologie et Pharmacologie Moléculaire (INSERM U573), Centre Paul Broca, 2 ter rue d'Alésia, 75014 Paris, France.
Stéphanie
S
Voisin
Didier
D
Rognan
Claude
C
Gros
Tanja
T
Ouimet
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003460:Crystallization; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000737:chemistry*; D051381:Rats
D008666:Metalloendopeptidases; C420382:NL1 metallopeptidase; D015260:Neprilysin; C476216:neprilysin 2, rat