Abstract
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.
Highlights
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
The model of the active site of NEP2 being based on the alignment of the C-terminal domains of model and template applies to both isoforms of NEP2 on one hand as well as to rNEP2 and human NEP2 on the other hand, because both active sites align perfectly (100% sequence identity, data not shown)
Coordinates used for NEP2 correspond to rNEP2(s), because this isoform was used in the site-directed mutagenesis studies
Summary
Materials—The synthetic fluorogenic substrate Suc-AAF-AMC, dipeptides and related amidated dipeptides, and biological peptides were purchased from Bachem (Voisins le Bretonneux, France). The three-dimensional model of NEP2 in complex with phosphoramidon was constructed by mutating the side chains of the amino acids in the hNEP-phosphoramidon structure (Protein Data Bank code 1dmt) to the respective side chains in NEP2. The insertions/deletions in the loops were achieved through a simple knowledgebased loop search procedure using the LOOPSEARCH module of the SYBYL package (TRIPOS Associates, Inc) In this procedure, a set of 1478 high resolution x-ray structures was searched for loops of similar length and similar distance between the C␣ atoms of the residues delimiting the loop window. For Km determinations, the incubation mixtures were comprised of 20 –50 g of the concentrated conditioned media and from 7.8 to 1000 M Suc-AAF-AMC in 100 l of 100 mM Hepes buffer, pH 7.2, containing 0.15 M NaCl and 0.01% Triton X-100. Statistical Analysis—The Student’s t tests were performed using the Instat program
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