Polar residues drive association of polyleucine transmembrane helices.

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161-166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154-160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Substrate-induced exposure of an energy-coupling motif of a membrane transporter.

BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli. It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in