PMID: 18303860

Storm J, Modok S, O'Mara ML, Tieleman DP, Kerr ID, Callaghan R
Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling.
Biochemistry. 2008 Mar 25;47(12):3615-24. Epub 2008 Feb 28., 2008-03-25 [PubMed]
Sentences
No. Mutations Sentence Comment
52 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:52:74
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:52:67
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:52:53
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:52:60
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:52:99
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:52:88
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 18303860:52:81
status: NEW
view ABCB1 p.Ser349Cys details
Single cysteine containing mutant isoforms of ABCB1 (S344C, V345C, G346C, Q347C, S349C, A354C, and G360C) were constructed as previously described (44) using site directed mutagenesis with the Altered Sites II (Promega) or the QuickChange (Stratagene) systems. Login to comment
114 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:114:72
status: NEW
view ABCB1 p.Val345Cys details
Figure 1a shows a representative example for the efficiency of labeling V345C with CM in the basal conformation. Login to comment
115 ABCB1 p.Gly324Cys
X
ABCB1 p.Gly324Cys 18303860:115:52
status: NEW
view ABCB1 p.Gly324Cys details
The positive control lane refers to labeling of the G324C isoform for 300 min and was assigned a value of 100%. Login to comment
116 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:116:81
status: NEW
view ABCB1 p.Val345Cys details
The data were quantified by densitometry and expressed graphically in Figure 1b. V345C was labeled fully (Lext ) 112%) with a half-life for the reaction of t½ ) 47 min. Login to comment
117 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:117:26
status: NEW
view ABCB1 p.Gly346Cys details
In contrast, labeling of G346C with FM did not reach a significant extent (Lext ) 7%), even after a 300 min incubation (Figure 1a,b). Login to comment
118 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:118:95
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:118:105
status: NEW
view ABCB1 p.Ala354Cys details
A complete graphical representation of similar labeling time-courses of two selected isoforms (V345C and A354C) is shown in Figure 2. Login to comment
120 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:120:17
status: NEW
view ABCB1 p.Val345Cys details
In contrast, the V345C isoform was highly accessible to CM, with complete labeling (Lext ) 111 ( 19% P < 0.05) observed during the course of the reaction and partially accessible to the zwitterionic BM probe (Lext ) 48 ( 6%). Login to comment
121 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:121:8
status: NEW
view ABCB1 p.Val345Cys details
Isoform V345C remained nonamenable to labeling with the hydrophilic FM when converted to the nucleotide bound or vanadate trapped conformation. Login to comment
122 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:122:9
status: NEW
view ABCB1 p.Val345Cys details
Trapping V345C in these two conformations also failed to impact significantly on the extent of labeling observed under basal conditions for either BM or CM (Figure 2). Login to comment
124 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:124:21
status: NEW
view ABCB1 p.Ala354Cys details
By contrast, isoform A354C (Figure 2) displays conformation dependent labeling of probes. Login to comment
128 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:128:15
status: NEW
view ABCB1 p.Ala354Cys details
BM labeled the A354C isoform equally well in the basal (Lext ) 99 ( 15%) and nucleotide bound conformations (Lext ) 130 ( 11%). Login to comment
134 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:134:151
status: NEW
view ABCB1 p.Ser344Cys details
Two striking features are evident from an examination of the data presented in the table, namely, a "polarity" of accessibility along the stretch from S344C FIGURE 1: Labeling of single cysteine isoforms of ABCB1 by maleimides. Login to comment
135 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:135:30
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:135:8
status: NEW
view ABCB1 p.Val345Cys details
(a) The V345C (upper gel) and G346C (lower gel) isoforms were reacted with CM and FM, respectively, for 10-300 min as described in Materials and Methods. Login to comment
137 ABCB1 p.Gly324Cys
X
ABCB1 p.Gly324Cys 18303860:137:41
status: NEW
view ABCB1 p.Gly324Cys details
ABCB1 p.Gly324Cys
X
ABCB1 p.Gly324Cys 18303860:137:205
status: NEW
view ABCB1 p.Gly324Cys details
The "+ve" lane refers to labeling of the G324C isoform for 300 min (b) The SDS-PAGE data was quantified using densitometric analysis, and the values were expressed as a percentage of that obtained for the G324C isoform. Login to comment
139 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:139:95
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:139:41
status: NEW
view ABCB1 p.Val345Cys details
Filled circles represent the labeling of V345C with CM, open circles represent the labeling of G346C with FM. Login to comment
141 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:141:66
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:141:80
status: NEW
view ABCB1 p.Ala354Cys details
The maximal extent of labeling (Lext) was ascertained for the (a) V345C and (b) A354C mutant isoforms of ABCB1 using the three maleimide containing probes, FM, BM and CM. Login to comment
144 ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:144:11
status: NEW
view ABCB1 p.Gly360Cys details
through to G360C and a disparity in the conformation dependent changes in accessibility. Login to comment
151 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:151:60
status: NEW
view ABCB1 p.Ala354Cys details
Similarly, large changes in accessibility are observed with A354C (Figure 2). Login to comment
155 ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 18303860:155:81
status: NEW
view ABCB1 p.Ser349Cys details
CM was chosen since it labels each of the chosen residues, with the exception of S349C (data not shown). Login to comment
158 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:158:71
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:158:61
status: NEW
view ABCB1 p.Gly346Cys details
The data in Table 2 confirms previous findings (44) that the G346C and Q347C mutations provided the greatest impairment to ABCB1 activity with the underlying defect caused by impaired basal activity. Login to comment
161 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:161:150
status: NEW
view ABCB1 p.Ala354Cys details
While there were no effects on the basal ATPase activity for any of the mutant isoforms, except for a 2-fold increase in the basal ATPase activity of A354C following CM labeling (Table 2), there were numerous distinct effects on the maximal extent of drug stimulated ATPase activity. Login to comment
163 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:163:66
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:163:76
status: NEW
view ABCB1 p.Val345Cys details
Representative full dose-response data is shown for two isoforms (S344C and V345C) in Figure 3. Login to comment
164 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:164:0
status: NEW
view ABCB1 p.Ser344Cys details
S344C displayed a significant reduction in the Vmax and degree of stimulation of ATP hydrolysis following the attachment of the coumarin moiety. Login to comment
166 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:166:416
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:166:366
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:166:255
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:166:313
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:166:574
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:166:517
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 18303860:166:467
status: NEW
view ABCB1 p.Ser349Cys details
A more dramatic effect was observed in this isoform following CM modification, namely, that vinblastine could no longer stimulate the basal ATPase Table 1: Summary of Relative Accessibilities of TM6 Residuesa ABCB1 isoform catalytic intermediate FM BM CM S344C basal - +++ +++ AMPPNP + +++ +++ vanadate + +++ +++ V345C basal - + +++ AMPPNP + ++ +++ vanadate + + +++ G346C basal - + ++ AMPPNP - ++ ++ vanadate + + ++ Q347C basal + + +++ AMPPNP + + ++ vanadate + + +++ S349C basal - + + AMPPNP + ++ + vanadate - ++ +++ A354C basal + +++ +++ AMPPNP ++ +++ +++ vanadate + + +++ G360C basal +++ +++ ++ AMPPNP +++ +++ ++ vanadate + ++ +++ a The accessibility of each introduced cysteine residue was determined using fluorescein-maleimide (FM), BODIPY-maleimide (BM) or coumarin-maleimide (CM). Login to comment
170 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:170:357
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:170:311
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:170:210
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:170:258
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:170:455
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:170:403
status: NEW
view ABCB1 p.Ala354Cys details
Table 2: Effects of Covalent Modification by CM on ATPase Activity of Mutant ABCB1 TM6 Isoformsa basal Vmax (nmol Pi min-1 mg-1) stimulated Vmax (nmol Pi min-1 mg-1) (-)CM (+) CM % change (-)CM (+) CM % change S344C 188 ( 62 192 ( 56 857 ( 216 317 ( 93* -63 V345C 563 ( 85 388 ( 41 -31 1173 ( 355 937 ( 292 -20 G346C 101 ( 18 102 ( 12 268 ( 47 186 ( 59 -30 Q347C 66 ( 6 41 ( 15 -37 161 ( 33 61 ( 4* -62 A354C 126 ( 10 229 ( 27* +81 535 ( 72 353 ( 18* -34 G360C 396 ( 82 508 ( 134 +28 1244 ( 252 810 ( 108 -34 a The ABCB1 isoforms containing mutations within TM6 were examined for ATPase activity prior to and following reaction with the hydrophobic maleimide probe CM. Login to comment
175 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:175:321
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:175:289
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:175:202
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:175:243
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:175:392
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:175:353
status: NEW
view ABCB1 p.Ala354Cys details
Table 3: Effects of Coumarin Labeling on the Potency of Drugs to Stimulate ATP Hydrolysis by Mutant ABCB1 TM6 Isoformsa EC50 nicardipine (µM) EC50 vinblastine (µM) (-) CM (+) CM (-) CM (+) CM S344C 3.0 ( 0.4 0.5 ( 0.1* 12.1 ( 0.5 na V345C 2.3 ( 0.4 3.9 ( 0.6 4.9 ( 1.4 8.5 ( 1.9 G346C 4.8 ( 1.8 7.1 ( 1.7 na na Q347C 2.7 ( 0.4 1.9 ( 0.8 na na A354C 2.4 ( 0.2 1.6 ( 0.2 2.5 ( 0.2 na G360C 2.7 ( 0.4 5.1 ( 0.2* 5.7 ( 0.1 8.6 ( 1.2 a The potency of nicardipine and vinblastine to stimulate ATP hydrolysis was examined for a wide range of drug concentrations (10-9-10-4 M). Login to comment
181 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:181:134
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:181:67
status: NEW
view ABCB1 p.Val345Cys details
Covalent modification also impacted the ATPase activity of isoform V345C, albeit in a markedly distinct fashion to that observed with S344C (Table 2 and Figure 3c and d). Login to comment
182 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:182:83
status: NEW
view ABCB1 p.Val345Cys details
The basal and nicardipine stimulated Vmax values for ATP hydrolysis by CM modified V345C were indistinguishable from that observed with untreated protein (Table 2). Login to comment
186 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:186:141
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 18303860:186:10
status: NEW
view ABCB1 p.Gly346Cys details
While the G346C isoform was unaffected by the reaction with CM, the Vmax for nicardipine stimulated ATP hydrolysis was reduced 2-fold in the Q347C isoform (Table 2). Login to comment
187 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:187:0
status: NEW
view ABCB1 p.Ala354Cys details
A354C, which was avidly labeled by CM, displayed a both small reduction in the Vmax for nicardipine stimulation of ATPase activity due to a reduced degree of stimulation by this compound. Login to comment
189 ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:189:0
status: NEW
view ABCB1 p.Gly360Cys details
G360C was less amenable to labeling with CM and only displayed a minor reduction in the potency of nicardipine to stimulate hydrolysis. Login to comment
194 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:194:20
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:194:13
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:194:31
status: NEW
view ABCB1 p.Ala354Cys details
The isoforms S344C, Q347C, and A354C displayed significant reductions in drug stimulated ATP hydrolysis following CM labeling, and this may be attributed to altered drug binding. Login to comment
200 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:200:37
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:200:57
status: NEW
view ABCB1 p.Val345Cys details
ATPase activity was obtained for the S344C (a and b) and V345C isoforms (c and d) of ABCB1 prior to (empty symbols) or following the reaction with CM (filled symbols). Login to comment
204 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:204:114
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:204:132
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:204:368
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:204:108
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:204:126
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:204:361
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:204:120
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:204:138
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:204:378
status: NEW
view ABCB1 p.Ala354Cys details
Table 4: Effects of Coumarin Labeling on Drug Binding in Mutant ABCB1 TM6 Isoformsa vinblastine nicardipine S344C Q347C A354C S344C Q347C A354C (-) CM 0.34 ( 0.09 0.39 ( 0.06 0.61 ( 0.11 0.35 ( 0.02 0.40 ( 0.03 0.64 ( 0.09 (+) CM 0.46 ( 0.06 0.34 ( 0.09 0.39 ( 0.21 0.27 ( 0.03 0.41 ( 0.06 0.57 ( 0.16 a [125I]-IAAP photoaffinity labeling was undertaken in the S344C, Q347C and A354C mutant TM6 isoforms prior to and following labeling with coumarin maleimide. Login to comment
212 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 18303860:212:66
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:212:59
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:212:77
status: NEW
view ABCB1 p.Ala354Cys details
Following labeling, three of the residues we have studied (S344C, Q347C, and A354C) were associated with a reduction in the magnitude to which drug substrates stimulate ATP hydrolysis. Login to comment
213 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:213:135
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:213:142
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:213:167
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:213:156
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 18303860:213:149
status: NEW
view ABCB1 p.Ser349Cys details
In addition, an alteration in the potency of the stimulation, or abrogation of ATPase activity, was observed for a number of residues (S344C, V345C, S349C, A354C, and G360C). Login to comment
234 ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 18303860:234:66
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:234:56
status: NEW
view ABCB1 p.Ala354Cys details
The observations were also undertaken for two residues (A354C and G360C) within the extension of TM6 that links the helix to the N-terminal NBD. Login to comment
237 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:237:21
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 18303860:237:31
status: NEW
view ABCB1 p.Ser349Cys details
The region including V345C and S349C was relatively intransigent to covalent modification with either FM or BM. Login to comment
239 ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 18303860:239:130
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Val331Cys
X
ABCB1 p.Val331Cys 18303860:239:121
status: NEW
view ABCB1 p.Val331Cys details
This lack of change is in contrast to the considerable conformational changes previously observed in the segment between V331C to F343C (42). Login to comment
240 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 18303860:240:8
status: NEW
view ABCB1 p.Ser344Cys details
Residue S344C, which is located in the central region of TM6, was considerably more accessible to covalent modification than the more cytosolic region of the helix. Login to comment
241 ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 18303860:241:127
status: NEW
view ABCB1 p.Phe343Cys details
This is in good agreement with the data from Rothnie et al. demonstrating similar high accessibility for the adjacent residue, F343C (42). Login to comment
242 ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 18303860:242:55
status: NEW
view ABCB1 p.Ser349Cys details
The residue at the proposed cytosolic terminus of TM6 (S349C) displayed the greatest propensity for changes in accessibility, indicating conformational flexibility in this region of the helix. Login to comment
249 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:249:215
status: NEW
view ABCB1 p.Val345Cys details
In silico mutation of the -branched valine residue to a slightly smaller cysteine residue, while less favorable in terms of residue packing, did not have a significant impact on the interhelical interactions of the V345C/L225/I306 triad. Login to comment
250 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:250:26
status: NEW
view ABCB1 p.Val345Cys details
The modeled side chain of V345C faces away from the pore in the basal state model, rendering it inaccessible to the hydrophilic FM probe and only partially accessible to the zwitterionic BM probe, in accordance with the data presented here (Figure 4a and Table 1). Login to comment
251 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:251:180
status: NEW
view ABCB1 p.Val345Cys details
Analysis of the AMP-PNP state model (which exhibits a "nucleotide sandwich dimer") suggests that a set of coordinated rotational and tilt angle changes in TM4, 5, and 6 around the V345C/L225/I306 triad occurs without altering the exposure of residue 345 itself (Figure 4b). Login to comment
254 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:254:39
status: NEW
view ABCB1 p.Ala354Cys details
Similar analysis helps rationalize the A354C data and provides further evidence that our homology models are a valid reflection of TM helix packing. Login to comment
256 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:256:0
status: NEW
view ABCB1 p.Ala354Cys details
A354C forms part of the TM3-TM6 contact interface with the sulfur atom being accessible to the translocation pore, thus enabling covalent modification by the hydrophilic FM and zwitterionic BM probes. Login to comment
257 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:257:48
status: NEW
view ABCB1 p.Ala354Cys details
Further examination of the residues adjacent to A354C revealed a number of charged and polar residues in its immediate vicinity, in particular, K181, E184, and D188 from TM3, and the adjacent E353 from TM6, creating a favorable local environment for FM and BM. Login to comment
258 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:258:116
status: NEW
view ABCB1 p.Ala354Cys details
Comparison of the AMP-PNP state model with the basal state model shows a disruption of the TM3-TM6 interface around A354C (Figure 4c). Login to comment
259 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:259:60
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:259:229
status: NEW
view ABCB1 p.Ala354Cys details
In the basal conformation homology model, the side chain of A354C was angled away from the pore, while charged residues in the local vicinity faced into the translocation pore (Figure 4d), decreasing the surface accessibility of A354C to the translocation pore and thus the accessibility to FM. Login to comment
260 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:260:16
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:260:136
status: NEW
view ABCB1 p.Ala354Cys details
The position of A354C at the interface of TM3 and TM6, which both have a lipid exposed face, allowed the hydrophobic CM probe to access A354C through the bilayer. Login to comment
261 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:261:163
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:261:173
status: NEW
view ABCB1 p.Ala354Cys details
On the basis of the observations in the present investigation and from previous studies (42-44), it appears that TM6 FIGURE 4: Molecular modeling insight into the V345C and A354C mutations in ABCB1. Login to comment
263 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:263:36
status: NEW
view ABCB1 p.Val345Cys details
(a) In the AMP-PNP bound state, the V345C (spacefill, CPK coloring) is inaccessible to the translocation pore. Login to comment
265 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:265:52
status: NEW
view ABCB1 p.Val345Cys details
The closest point of contact between the helices is V345C, which interacts hydrophobically with Leu225 (orange) and Ile306 (green). Login to comment
266 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:266:68
status: NEW
view ABCB1 p.Val345Cys details
(b) The modeled basal state shows only minor reorganizations of the V345C-L225-I306 triad, consistent with the experimental findings. Login to comment
267 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:267:69
status: NEW
view ABCB1 p.Ala354Cys details
(c) A view from the top of the pore shows the AMP-PNP configuration: A354C (orange) hydrogen bonds to its nearest neighbour, D188 (cyan). Login to comment
268 ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 18303860:268:104
status: NEW
view ABCB1 p.Val345Cys details
(d) In the basal state, conformational changes disrupt the TM3-TM6 coiled-coil motif and break the D188-V345C hydrogen bond. Login to comment
269 ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 18303860:269:94
status: NEW
view ABCB1 p.Ala354Cys details
Adjacent charged residues from TM3 (cyan) and TM6 (purple) face the translocation pore, while A354C is situated in a cleft between TM3 and TM6. Login to comment