ABCMdb

PMID: 18658148

He L, Aleksandrov AA, Serohijos AW, Hegedus T, Aleksandrov LA, Cui L, Dokholyan NV, Riordan JR
Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating.
J Biol Chem. 2008 Sep 26;283(39):26383-90. Epub 2008 Jul 25., 2008-09-26 [PubMed]

Sentences

No. Mutations Sentence Comment

75 ABCC7 p.Tyr1307Cys To verify these specific interactions, we performed cross-linking experiments in HEK cells expressing a Cys-less CFTR construct containing the Cys pair 276C and Y1307C (Fig. 2A). Login to comment 80 ABCC7 p.Leu1065Cys ABCC7 p.Trp496Cys ABCC7 p.Thr1064Cys ABCC7 p.Met498Cys In agreement with what is predicted by our model, we were able to cross-link residue pairs W496C/T1064C, M498C/L1065C (19), which proves that indeed CL4 also interacts with NBD1 through the so called Q-loop (Q493). Login to comment 84 ABCC7 p.Asn268Cys ABCC7 p.Phe1294Cys The successful cross-linking of the residue pairs N268C (CL2) and F1294C (NBD2) by the MTS reagents of various spacer arm lengths confirmed this contact (Fig. 2A). Login to comment 90 ABCC7 p.Asn268Cys ABCC7 p.Asp1341Cys ABCC7 p.Thr1057Cys ABCC7 p.Thr966Cys ABCC7 p.Glu543Cys ABCC7 p.Leu172Cys Cys pair cross-linking experiments showed that indeed E543C could be cross-linked with both T966C (CL3) and T1057C (CL4, Fig. 2B), while D1341C was in close contact with both L172C (CL1) and N268C (CL2, Fig. 2C). Login to comment 100 ABCC7 p.Tyr1307Cys ABCC7 p.Asn268Cys A, 276C/Y1307C and N268C/F1292C at the CL2/NBD2 interface involving an aromatic cluster and the Q-loop. Login to comment 101 ABCC7 p.Thr1057Cys ABCC7 p.Thr966Cys ABCC7 p.Glu543Cys ABCC7 p.Glu543Cys B, T966C/E543C at the CL3/NBD1 interface and T1057C/E543C at the CL4/NBD1 interface at the X-loop of NBD1. Login to comment 102 ABCC7 p.Asn268Cys ABCC7 p.Asp1341Cys ABCC7 p.Asp1341Cys ABCC7 p.Leu172Cys C, L172C/D1341C at the CL1/NBD2 interface and N268C/D1341C at the CL2/NBD2 interface at the X-loop of NBD2. Login to comment 106 ABCC7 p.Leu1260Cys ABCC7 p.Leu1261Cys ABCC7 p.Met961Cys ABCC7 p.Ser962Cys To confirm these contacts in the case of CL3 and NBD2, we designed several Cys pairs from CL3 (M961C and S962C) and NBD2 (L1260C and L1261C) (Fig. 3A). Login to comment 108 ABCC7 p.Leu1260Cys ABCC7 p.Leu1261Cys ABCC7 p.Leu1261Cys ABCC7 p.Met961Cys ABCC7 p.Met961Cys ABCC7 p.Ser962Cys In constructs containing the Cys pairs M961C/L1261C (Fig. 3A), M961C/L1260C, and S962C/ L1261C (supplemental Fig. S1A), MTS reagent treatment produced a slightly, albeit distinguishably, faster moving band, which could be reversed by DTT. Login to comment 111 ABCC7 p.Leu1261Cys ABCC7 p.Met961Cys Cross-linking of Cys pairs between CL3 and NBD2 was also confirmed with co-expression of constructs of Cys-less ⌬NBD2 CFTR containing M961C together with the Cys-less NBD2 fragment containing L1261C in HEK cells (supplemental Fig. S1, B and C). Login to comment 116 ABCC7 p.Glu407Cys ABCC7 p.Val171Cys ABCC7 p.Val171Cys ABCC7 p.Leu408Cys Based on the latter model, we attempted to detect cross-linking of the residue pairs V171C/E407C and V171C/L408C in the whole protein, but did not observe any indication of cross-linking of the mature band (supplemental Fig. S2A). Login to comment 119 ABCC7 p.Val171Cys ABCC7 p.Leu408Cys To test this hypothesis, membrane vesicles were prepared from BHK cells overexpressing Cys-less CFTR containing V171C and L408C. Login to comment 125 ABCC7 p.Glu407Cys ABCC7 p.Val171Cys We found similar results for the Cys pair V171C and E407C (supplemental Fig. 2B), but not with a Cys pair introduced at Gln-958 and Leu-1261 (supplemental Fig. S2C), confirming that the faster moving CFTR fragment was indeed due to the cross-linking at the CL1/NBD1 interface. Login to comment 127 ABCC7 p.Asp1341Cys ABCC7 p.Thr966Cys ABCC7 p.Glu543Cys ABCC7 p.Leu172Cys ABCC7 p.Leu1261Cys ABCC7 p.Met961Cys ABCC7 p.Val171Cys ABCC7 p.Leu408Cys No cross-linking was detected when Cys pairs were introduced at L172C/E543C, T966C/D1341C, V171C/L1261C, or M961C/L408C, which are not predicted to be in association in the structural model (supplemental Fig. S3). Login to comment 135 ABCC7 p.Val171Cys ABCC7 p.Leu408Cys Membrane vesicles prepared from BHK cells overexpressing Cys-less CFTR containing the Cys pair V171C and L408C were treated with 20 ␮M MTS cross-linker M8M before limited trypsin digestion with concentrations indicated in the figures. Login to comment 142 ABCC7 p.Thr1057Cys ABCC7 p.Thr966Cys ABCC7 p.Glu543Cys ABCC7 p.Glu543Cys To determine whether the interfacial interaction of Glu-543 with CL3 and CL4 was indeed affected by PKA phosphorylation, membrane vesicles from HEK cells overexpressing Cys-less CFTR with Cys pairs E543C/T966C and E543C/T1057C were pretreated with PKA in the presence of ATP and Mg2ϩ before cross-linking with various MTS reagents. Login to comment 143 ABCC7 p.Thr1057Cys ABCC7 p.Thr966Cys ABCC7 p.Glu543Cys ABCC7 p.Glu543Cys As shown in Fig. 4A, similar to the experiments with whole cells in membranes not treated with PKA, the Cys pairs E543C/T966C and E543C/T1057C were cross-linked by all the MTS reagents tested. Login to comment 150 ABCC7 p.Met498Cys ABCC7 p.Asn268Cys ABCC7 p.Phe1294Cys ABCC7 p.Gly1061Cys As shown in Fig. 5, treatment of the cells with PKA stimulation mixture before and during cross-linking had no effect on cross-linking between either M498C/G1061C (Fig. 5A, left panel) or N268C/F1294C (Fig. 5B, left panel), although phosphorylation-sensitive CFTR antibody mAb 450 confirmed the phosphorylation of CFTR (Fig. 5, A and B, right panels). Login to comment 151 ABCC7 p.Trp496Cys ABCC7 p.Lys1292Cys However, when a Cys mutation was introduced in each Q-loop (W496C/K1292C), the PKA stimulation mixture significantly increased the cross-linking of this Cys pair with all MTS reagents tested (Fig. 5C). Login to comment 156 ABCC7 p.Leu1261Cys ABCC7 p.Met961Cys First, cross-linking between residues M961C and L1261C at the CL3/ NBD2 interface changed channel gating behavior substantially but did not arrest it completely (Fig. 6A). Login to comment 159 ABCC7 p.Thr1064Cys ABCC7 p.Thr966Cys ABCC7 p.Glu543Cys Membrane vesicles prepared from HEK cells transiently transfected with Cys pairs introduced at E543C with T966C (CL3) and T1064C (CL4) were pretreated with PKA catalytic subunit in the presence of ATP before incubating with 20 ␮M MTS reagents. Login to comment 166 ABCC7 p.Trp496Cys ABCC7 p.Met498Cys ABCC7 p.Asn268Cys ABCC7 p.Phe1294Cys ABCC7 p.Gly1061Cys ABCC7 p.Lys1292Cys Cys pairs were introduced at: A interface between NBD1 Q-loop and CL4 (M498C/G1061C); B, interface between NBD2 Q-loop and CL2 (N268C/ F1294C); or C, both Q-loops (W496C and K1292C). Login to comment 170 ABCC7 p.Glu543Cys In contrast, the cross-linking between residues T996C and E543C at the CL3/NBD1 interface rapidly and reversibly arrested channel gating (Fig. 6B) exactly as we had observed previously with the CL4/NBD1 interface (19). Login to comment 172 ABCC7 p.Val171Cys ABCC7 p.Leu408Cys Turning from the effects of cross-linking CL3 to those of its counterpart, CL1 in the N-terminal MSD, its cross-linking to NBD1 i.e. V171C/L408C had essentially no influence on gating (Fig. 6C). Login to comment 173 ABCC7 p.Asp1341Cys ABCC7 p.Leu172Cys On the other hand, the cross-linking between L172C of CL1 and D1341C of NBD2 rapidly and reversibly inhibited channel gating (Fig. 6D) very similar to what we had observed previously for the CL2/NBD2 interface (19). Login to comment 174 ABCC7 p.Asp1341Cys ABCC7 p.Leu172Cys However, in the case of the completely reversible cessation of gating caused by L172C/D1341C cross-linking, the role of modification of each of these cysteines individually will have to be studied in detail because treatment with monofunctional MTSES also inhibited gating. Login to comment 203 ABCC7 p.Asp1341Cys ABCC7 p.Thr966Cys ABCC7 p.Glu543Cys ABCC7 p.Leu1261Cys ABCC7 p.Met961Cys ABCC7 p.Val171Cys ABCC7 p.Leu408Cys A, M961C/L1261C at the CL3/NBD2 interface; B, T966C/E543C at the CL3/NBD1 interface; C, V171C/L408C at the CL1/NBD1 interface; D, L171C/D1341C at the CL1/NBD2 interface. Login to comment 222 ABCC7 p.Ser909Cys In P-glycoprotein, the trapping of AMP-PNP or ADP plus vanadate at NBD reduces the cross-linking of L443C and S909C, suggesting that conformational changes occur at the NBD1/MSD2 interface during the ATP catalytic cycle (33). Login to comment 244 ABCC7 p.Glu407Cys ABCC7 p.Val171Cys ABCC7 p.Val171Cys ABCC7 p.Leu408Cys We have verified the latter conformation by introducing Cys pairs in CL1 and different residues in RI (V171C/ E407C, V171C/L408C) and showing that these pairs can be cross-linked by M8M (Fig. 3B). Login to comment