title: Analysis of putative protomer crosstalk in the trimeric transporter BetP: The heterotrimer approach.
abstract: The homotrimeric, secondary active betaine carrier BetP from Corynebacterium glutamicum is a model system for stress-regulated transport in bacteria. Its activity responds to hyperosmotic stress and it harbors two different functions, transport catalysis (betaine uptake) and stimulus sensing, resp. activity regulation. Structural information from 2D and 3D crystals as well as functional analysis of monomerized BetP suggested the presence of conformational crosstalk between the individual protomers. To study whether the oligomeric state is functionally significant on a mechanistic level we generated heterooligomeric complexes of BetP in which single protomers within the trimer can be addressed. By testing dominant negative effects in a trimer of one active protomer combined with two protomers in which transport and regulation were abolished, we provide experimental evidence for the absence of functionally significant conformational crosstalk between the protomers on the level of both transport and regulation. This is supported by experiments using mutant forms of putative interacting signal donor and acceptor domains of individual BetP protomers. This result has important consequences for oligomeric transport proteins in general and BetP in particular.
authors.affiliation: Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Becker
authors.affiliation: Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Maximov
authors.affiliation: Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Becker
authors.affiliation: Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Meyer
authors.affiliation: Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Wittmann
authors.affiliation: Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany. Electronic address: r.kraemer@uni-koeln.de.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Krämer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001704:Biopolymers / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D011487:Protein Conformation; D027981:Symporters
chemical_list: D001426:Bacterial Proteins; C101652:BetP protein, Corynebacterium glutamicum; D001704:Biopolymers; D002352:Carrier Proteins; D027981:Symporters
keywords: BetP; Oligomerization; Osmoregulation; Protein interaction; Transport