title: Studies of recombinant TWA1 reveal constitutive dimerization.
abstract: The mammalian muskelin/RanBP9/C-terminal to LisH (CTLH) complex and the Saccharomyces cerevisiae glucose-induced degradation (GID) complex are large, multi-protein complexes that each contain a RING E3 ubiquitin ligase. The yeast GID complex acts to degrade a key enzyme of gluconeogenesis, fructose 1,6-bisphosphatase, under conditions of abundant fermentable carbon sources. However, the assembly and functions of the mammalian complex remain poorly understood. A striking feature of these complexes is the presence of multiple proteins that contain contiguous lissencephaly-1 homology (LisH), CTLH and C-terminal CT11-RanBP9 (CRA) domains. TWA1/Gid8, the smallest constituent protein of these complexes, consists only of LisH, CTLH and CRA domains and is highly conserved in eukaryotes. Towards better knowledge of the role of TWA1 in these multi-protein complexes, we established a method for bacterial expression and purification of mouse TWA1 that yields tag-free, recombinant TWA1 in quantities suitable for biophysical and biochemical studies. CD spectroscopy of recombinant TWA1 indicated a predominantly α-helical protein. Gel filtration chromatography, size-exclusion chromatography (SEC) with multi-angle light scattering (SEC-MALS) and native PAGE demonstrated a propensity of untagged TWA1 to form stable dimers and, to a lesser extent, higher order oligomers. TWA1 has a single cysteine residue, Cys139, yet the dimeric form was preserved when TWA1 was purified in the presence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). These findings have implications for understanding the molecular role of TWA1 in the yeast GID complex and related multi-protein E3 ubiquitin ligases identified in other eukaryotes.
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K.
authors.forename: Ore
authors.initials: O
authors.lastname: Francis
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K.
authors.forename: Genevieve E
authors.initials: GE
authors.lastname: Baker
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K.|BrisSynBio Synthetic Biology Research Centre, University of Bristol, Bristol BS8 1TQ, U.K.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Race
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K. jo.adams@bristol.ac.uk.
authors.forename: Josephine C
authors.initials: JC
authors.lastname: Adams
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D000072756:Protein Conformation, alpha-Helical; D000072417:Protein Domains; D055503:Protein Multimerization; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; C000625574:Twa1 protein, mouse
keywords: CTLH; LisH; TWA1; Ubiquitin; protein purification