Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein

Abstract

TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim. Biophys. Acta 1798(11), 2160-2169 (2010)] that TrwB can be reconstituted into liposomes, and that bilayer incorporation increases its affinity for nucleotides and its specificity for ATP. In the present contribution we examine the structural effects of membrane insertion on TrwB, by comparing the protein in reconstituted form and in the form of protein/lipid/detergent mixed micelles. TrwB was reconstituted in PE:PG:CL (76.3:19.6:4.1mol ratio) with a final 99:1 lipid:protein mol ratio. This lipid mixture is intended to mimic the bacterial inner membrane composition, and allows a more efficient reconstitution than other lipid mixtures tested. The studies have been carried out mainly using infrared spectroscopy, because this technique provides simultaneously information on both the lipid and protein membrane components. Membrane reconstitution of TrwB is accompanied by a decrease in β-sheet contents and an increase in β-strand structures, probably related to protein-protein contacts in the bilayer. The predominant α-helical component remains unchanged. The bilayer-embedded protein becomes thermally more stable, and also more resistant to trypsin digestion. The properties of the bilayer lipids are also modified in the presence of TrwB, the phospholipid acyl chains are slightly ordered, and the phosphate groups at the interface become more accessible to water. In addition, we observe that the protein thermal denaturation affects the lipid thermal transition profile.

DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System

“…Finally, although we favor the notion that the periplasmic domain mutations disrupt a VirD4-VirB10 binding partner interface, we cannot exclude the possibility that the mutations exert their effects indirectly through global disruption of VirD4's conformational or oligomeric state. Extensive biochemical studies by the de la Cruz and Alkorta groups showing the importance of TrwB's N-terminal TMD to its activity, structure, and stability are consistent with such a possibility (68)(69)(70)(71)(72).…”

Towards an integrated model of bacterial conjugation

“…At the N-terminal domain of TrwB there are two transmembrane α-helices which anchor the protein to the inner membrane. This transmembrane domain also regulates nucleotide binding affinity (Vecino, et al, 2010) and it is important in stabilizing the structure of the protein (Vecino, et al, 2011).…”

The expanding bacterial type IV secretion lexicon

“…Genetic, biochemical, and structural evidence exists for T4CP binding to DNA as well as cognate relaxases and other components of the relaxosome (Atmakuri et al, 2007; Lu et al, 2008). Purified TrwB R388 exhibits DNA-stimulated ATPase and oligomerization activities (Tato et al, 2005), and is also stabilized by insertion into membrane lipids (Vecino et al, 2011). TrwB R388 reconstitution in liposomes enhances nucleotide binding affinity as well as ATP-specific binding (Vecino et al, 2010).…”