BACK TO DIAGRAM CFTR REVIEW PAGE
EXTRACELLULAR LOOP 2
is WELLQ (amino acids 216 thru 220)
In May, 2001, Hammerle et al. reported on a study
involving extracellular loop contributions to both maturation and chloride
activity. They stated that "The first and fourth
extracytoplasmic loops (ELs) contain approximately 15 and 30 residues,
respectively; the other four ELs are extremely short. To
examine the influence of missense mutants in ELs detected in patients with
cystic fibrosis, we have expressed them in mammalian (baby hamster kidney
(BHK21)) cells and assessed their biosynthetic processing and chloride channel
activity. In contrast to previous findings that 18 of 30
disease-associated missense mutations in cytoplasmic loops caused retention of
the nascent polypeptides in the endoplasmic reticulum, all the EL mutants
studied matured and were transported to the cell surface. This
pronounced asymmetry is consistent with the notion that endoplasmic reticulum
quality control of nascent CFTR is exerted primarily on the cytoplasmic side of
the membrane. Although this set of EL mutations has little effect on CFTR
maturation, most of them seriously compromise its chloride channel activity.
Substitutions at six different positions in EL1 and single positions in EL2 and
EL4 all destabilized the open state, some of them severely, indicating that the
ELs contribute to the stability of the CFTR ion pore."
J Biol Chem 2001 May 4;276(18):14848-54
In the crystal structure of MsbA, the extracellular loop 2 was found to be from residues 165-167, and it has strong polar character. Strong electron density was seen for Trp-165. Science 9/7/01, Vol 293 pgs 1793-1800
Transmembrane 4
ASAFCGLGFLIVLALFQAGLG
(amino acids 221 thru 241)
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. Biophysical Society Meeting, 2001
Partridge AW, et al of the Hospital for Sick Children in
Toronto,Canada reported in March 2002 the following: "Polar
side chains constitute over 20% of residues in the transmembrane (TM) helices of
membrane proteins, where they may serve as hydrogen bond interaction sites for
phenotypic polar mutations that arise in membrane protein-related
diseases. To systematically explore the structural
consequences of H-bonds between TM helices, we focused on TM4 of CFTR and its
cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model
system. Synthetic peptides corresponding to wild-type (TM4-wt)
(residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences
both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE
arising from disulfide bond formation via wild-type residue
Cys-225. However, the TM4-V232D peptide additionally forms a
ladder of noncovalent oligomers, including tetramers, hexamers, and octamers,
mediated by a hydrogen bond network involving Asp-Gln side chain-side chain
interactions. Ala-scanning mutagenesis of the TM4 sequence
indicated that ladder formation minimally required the simultaneous presence of
the Cys-225, Asp-232, and Gln-237 residues. As random
hydrophobic sequences containing these three residues at TM4 equivalent
positions did not oligomerize, specific van der Waals packing interactions
between helix side chains were also shown to play a crucial
role. Overall, the results suggest that polar mutations in
membrane domains, in conjunction with critically positioned polar partner
residues, potentially constitute a source of aberrant helix interactions that
could contribute to loss of function when they arise in protein transmembrane
domains." Biochemistry 2002 Mar 19;41(11):3647-53
Extracellular loop 3
K
(amino acid 329)
Transmembrane helix 7
I IFVLIWCLVIFLAEVAASLVV
(amino acids 860 thru 870)
Transmembrane helix 8
SYYVFYIYVGVADTLLAMGFF
(amino acids 912 thru 932)
Transmembrane helix 9
IFDFIQLLLIVIGAIAVVAVL
(amino acids 991 thru 1011)
Extracellular loop 5
QP
(amino acids 1012 and 1013)
Intracellular loop 5
QTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYL
STLRWFQMR
(amino acids 1035 thru 1102)
Transmembrane Helix 11
IEMIFVIFFIAVTFISILTTG
(amino acids 1103 thru 1123)
Extracellular loop 6
EGEGR
(amino acids 1124 to 1128)
Intracellular Loop 5 (which also includes NBD2: see NBD2 for that sequence, as well as the C-terminal sequence)
IDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTV
KDLTAK
(amino acids 1151 thru 1218)