title: Intersubunit and domain interactions of the meprin B metalloproteinase. Disulfide bonds and protein-protein interactions in the MAM and TRAF domains.
abstract: Meprins, multimeric metalloproteases expressed in kidney and intestinal epithelial cells as well as in certain leukocytes and cancer cells, have the ability to hydrolyze a variety of growth factors, vasoactive peptides, cytokines, and extracellular matrix proteins. The meprin B isoform exists primarily as a cell-surface homooligomer composed of disulfide-linked, multidomain beta-subunits. To gain insight into how the tertiary and quaternary structure of meprin B affects function, the disulfide-bonding pattern and sites of domain-domain interactions were investigated using sedimentation equilibrium ultracentrifugation, cross-linking, and mass spectrometry techniques. Three symmetrical intersubunit disulfide bonds were identified in the noncatalytic interaction domains; two in the MAM (meprin, A-5 protein, protein-tyrosine phosphatase mu) domain and one in the TRAF (tumor necrosis factor receptor-associated factor) domain. These disulfide bridges are unique for the known homophilic interactions of these domains. Mutation of any of the intersubunit cysteine residues resulted in the inability of meprin B to form disulfide-linked dimers. The four cysteines of the protease domain formed intradomain disulfide bonds. The MAM domain also had one intradomain disulfide bond and one free cysteine. Cross-linking studies of the meprin B dimer with the amine-reactive cross-linker disuccinimidyl suberate revealed inter- and intradomain contacts within the protein, including prosequence-prosequence, protease-TRAF, protease-epidermal growth factor, and TRAF-TRAF interactions. From these observations, a model of the meprin B dimer structure is proposed that provides insight into the relationship between structure and function of this isoform.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
authors.forename: Faoud T
authors.initials: FT
authors.lastname: Ishmael
authors.forename: Vincent K
authors.initials: VK
authors.lastname: Shier
authors.forename: Susan S
authors.initials: SS
authors.lastname: Ishmael
authors.forename: Judith S
authors.initials: JS
authors.lastname: Bond
mesh_terms: D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins; D013388:Succinimides; D008666:Metalloendopeptidases; C095663:meprin B; D003545:Cysteine; C019358:disuccinimidyl suberate