title: Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein.
abstract: Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The protein specificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity.
authors.affiliation: Departamento de Bioquímica, Universidad del País Vasco, 48080 Bilbao, Spain.
authors.forename: Ana J
authors.initials: AJ
authors.lastname: Vecino
authors.forename: Rosa L
authors.initials: RL
authors.lastname: Segura
authors.forename: Begoña
authors.initials: B
authors.lastname: Ugarte-Uribe
authors.forename: Sandra
authors.initials: S
authors.lastname: Aguila
authors.forename: Itsaso
authors.initials: I
authors.lastname: Hormaeche
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.forename: Itziar
authors.initials: I
authors.lastname: Alkorta
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D003227:Conjugation, Genetic; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry; D008051:Lipid Bilayers / Q000737:chemistry*; D009711:Nucleotides / Q000378:metabolism*; D011510:Proteolipids / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D009711:Nucleotides; D011510:Proteolipids; C020485:proteoliposomes; C403125:trwB protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases