BACK TO DIAGRAM CFTR REVIEW PAGE
TRANSMEMBRANE HELIX 3
LALAHFVWIAPLQVALLMGLI
(amino acids 195 thru 215)
Channel-lining water accessible residues have been found in transmembrane helices 1, 3, and 6 using the substituted cysteine accessibility method.
For information on pore, see helix1.
RECENT EVIDENCE: More Pieces to the Puzzle?
In December, 2001 Therien and Deber (from Sick Children's Hospital, Toronto, CA) reported making a helix-loop-helix construct consisting of the adjacent transmembrane segments 3 and 4 of the cystic fibrosis transmembrane conductance regulator (CFTR) labeled with pyrene at both N- and C-termini. Here, they describe a method for studying intramolecular helix-helix interactions within membrane proteins (i.e. CFTR). They measured pyrene excimer band intensity as a determinant of helix-helix proximity and showed that the helices retain tertiary contacts in detergent micelles. They state that this new method provides information about the "role of lipids on membrane protein folding and conformation, as well as the usefulness of a pyrene-based system in studying the forces that govern interhelical packing." J Biol Chem 2001 Dec 17
The only known high-resolution x-ray crystal structure of an ABC transporter family member is of MsbA. It reveals a central chamber formed within the membrane by contacts between transmembrane helices 2 and 5. This is due the the fact that MsbA, like all bacterial ABC transporters functions as a homodimer (while CFTR functions as a single protein), with TM2 of MsbA contacting TM5 in the opposite half of the protein. These four transmembrane helices can be seen as forming a kind of "hinge" (2 each) on either side of the chamber. All 6 of the transmembrane helices in the MsbA monomer are tilted with respect to the plane of the membrane by between 30 and 40 degrees. It has been predicted by the authors that during gating of MsbA, the transmembrane helices either flip or twist to expose newly accessible areas within the chamber. The portion of MsbA which corresponds to TM3 of CFTR is from amino acids 140-164. Chang and Roth, Science 293, 9/7/01 pgs 1793-1800.
Therlen and Deber (Hospital for Sick Children, Toronto, Canada) made constructs consisting of fused TM3-TM4 and found wild-type sequences migrated slower on SDS-PAGE and PFO-PAGE than a common CF mutant (V232D in TM4). They explained this difference in mobility on the gel by assuming the mutant V232D formed a new hydrogen bond between the mutant D232 (TM4) and Q207 (from TM3) because this could create a more compact hairpin loop. The conclusion being that a new interaction between the TM helices 3 and 4 occurs in the mutant CFTR. In their article in Nature Structural Biology, they write that "...such a hydrogen bond could alter the normal assembly and alignment of CFTR TM helices and/or impede their movement in response to substrate transport. Our results imply that membrane proteins may be vulnerable to loss of function through formation of membrane-buried interhelical hydrogen bonds by partnering of proximal polar side chains." Biophysical Society Meeting, 2001. Nat Struct Biol 2001 Jul;8(7):597-601
Wigley WC, et al from the University of Texas Southwestern Medical Center in
Dallas wrote in May 2002 that "the linear sequence of amino acids
contains all the necessary information for a protein to fold into its unique
three-dimensional structure. Native protein sequences are known to accomplish
this by promoting the formation of stable, kinetically accessible
structure. We describe a Pro residue in the center of the
third transmembrane helix of CFTR that promotes folding by a distinct mechanism:
disfavoring the formation of a misfolded structure. The
generality of this mechanism is supported by genome-wide transmembrane sequence
analyses. Furthermore, the results provide an explanation for the
increased frequency of Pro residues in transmembrane
alpha-helices. Incorporation by nature of such 'negative
folding determinants', aimed at preventing the formation of off-pathway
structures, represents an additional mechanism by which folding information is
encoded within the evolved sequences of proteins." Nat
Struct Biol 2002 May;9(5):381-8