Rational redesign of neutral endopeptidase binding to merlin and moesin proteins.
Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein-protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K(19)K(20)K(21) basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E(159)Q(160) in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N(159)L(160) residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N(159)L(160)Y(161)K(162)M(163) in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide-merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function.
Niv
Masha Y
MY
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
Iida
Katsuyuki
K
Zheng
Rong
R
Horiguchi
Akio
A
Shen
Ruoqian
R
Nanus
David M
DM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D003198:Computer Simulation; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015260:Neprilysin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D025581:Neurofibromin 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D008840:Microfilament Proteins; D025581:Neurofibromin 2; D010455:Peptides; C071058:moesin; D015260:Neprilysin