title: Membrane-enabled dimerization of the intrinsically disordered cytoplasmic domain of ADAM10.
abstract: Intrinsically disordered protein regions are widely distributed in the cytoplasmic domains of many transmembrane receptors. The cytoplasmic domain of a disintegrin and metalloprotease (ADAM)10, a transmembrane metalloprotease mediating ectodomain shedding of diverse membrane proteins, was recently suggested to mediate the homodimerization of ADAM10. Here we show that a recombinant cytoplasmic domain of ADAM10 (A10Cp) is unstructured as judged by its susceptibility to limited trypsin digestion and its circular dichroism spectrum. In comparison, recombinant transmembrane-cytoplasmic domain of ADAM10 (A10TmCp) reconstituted in dodecylphosphocholine (DPC) micelles exhibits much greater resistance to trypsin digestion, with its cytoplasmic domain taking on a significant ordered structure. FRET analysis demonstrates that, although A10Cp remains monomeric, A10TmCp forms a tight homodimer (K(d) ∼ 7 nM) in DPC micelles. Phospholipid-conjugated A10Cp dose-dependently inhibits formation of A10TmCp homodimer, whereas A10Cp achieves only limited inhibition. Placing the transmembrane and cytoplasmic domains of ADAM10, but not the transmembrane domain alone, in their native orientation in the inner membrane of Escherichia coli produces specific and strong dimerization signal in the AraC-based transcriptional reporter assay. A chimeric construct containing the otherwise monomeric transmembrane domain of L-selectin and the cytoplasmic domain of ADAM10 produces a similar dimerization signal. Overall, these results demonstrate that a transmembrane domain imparts a stable structure to the adjacent and intrinsically disordered cytoplasmic domain of ADAM10 to form a homodimer in the membrane. This finding advances our understanding of the regulatory mechanism of ADAMs and has general implications for membrane-protein interactions in the process of transmembrane signaling.
authors.affiliation: Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322; and.
authors.forename: Wei
authors.initials: W
authors.lastname: Deng
authors.affiliation: Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322; and.
authors.forename: Sungyun
authors.initials: S
authors.lastname: Cho
authors.affiliation: Department of Chemical Engineering, Lehigh University, Bethlehem, PA 18015.
authors.forename: Pin-Chuan
authors.initials: PC
authors.lastname: Su
authors.affiliation: Department of Chemical Engineering, Lehigh University, Bethlehem, PA 18015.
authors.forename: Bryan W
authors.initials: BW
authors.lastname: Berger
authors.affiliation: Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322; and renhao.li@emory.edu.
authors.forename: Renhao
authors.initials: R
authors.lastname: Li
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human
keywords: ADAM10; FRET; Intrinsic disorder; membrane protein dimerization