Characterization of the structure and intermolecular interactions between the connexin 32 carboxyl-terminal domain and the protein partners synapse-associated protein 97 and calmodulin.
In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca(2+), pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis.
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Kelly
K
Stauch
Fabien
F
Kieken
Paul
P
Sorgen
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074288:Discs Large Homolog 1 Protein; D017629:Gap Junctions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051528:Guanylate Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D012583:Schwann Cells / Q000166:cytology / Q000378:metabolism*; D000097002:Gap Junction beta-1 Protein
D048868:Adaptor Proteins, Signal Transducing; D002147:Calmodulin; D017630:Connexins; D000074288:Discs Large Homolog 1 Protein; D051528:Guanylate Kinases; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D000097002:Gap Junction beta-1 Protein; C000614189:Dlg1 protein, mouse; C501119:Dlg1 protein, rat