Lipid interactions enhance activation and potentiation of cystic fibrosis transmembrane conductance regulator (CFTR)

The recent cryo-electron microscopy structures of phosphorylated, ATP-bound CFTR in detergent micelles failed to reveal an open anion conduction pathway as expected on the basis of previous functional studies in biological membranes. We tested the hypothesis that interaction of CFTR with lipids is important for opening of its channel. Interestingly, molecular dynamics studies revealed that phospholipids associate with regions of CFTR proposed to contribute to its channel activity. More directly, we found that CFTR purified together with associated lipids using the amphipol: A8-35, exhibited higher rates of catalytic activity, channel activation and potentiation using ivacaftor, than did CFTR purified in detergent. Catalytic activity in CFTR detergent micelles was partially rescued by addition of phospholipids plus cholesterol, arguing that these lipids contribute directly to its modulation. In summary, these studies highlight the importance of lipids in regulated CFTR channel activation and potentiation.

and TM11. As yet, the molecular basis for the positive effect of POPS on CFTR ATPase activity 120 remains unknown (Hildebrandt, Khazanov et al. 2017), but our analyses support the hypothesis 121 that its interaction with positively charged residues lining the cleft between TM4 and TM6 may 122 disrupt previously described interactions with organic anions known to be inhibitory to CFTR 123 ATPase activity (Kogan, Ramjeesingh et al. 2001  Full-length human Wt-CFTR, bearing its native sequence except for two affinity tags, a FLAG 132 tag (on the amino terminus) and a 10x histidine-tag (on the carboxy terminus) was expressed in  Size exclusion chromatography (SEC) was used to characterize the purified CFTR protein on the 142 basis of differences in size using 280 nm to monitor the elution of CFTR (Figure 2b). The 143 fractions eluting between elution volume of 9-17 ml containing both core and complex 144 glycosylated CFTR, as confirmed by silver staining and immunoblotting, co-eluted with ATPase 145 activity ( Figure 2b). Interestingly, the peak ATPase activity at elution volume of 14.5 ml was 146 slightly displaced from the peak CFTR protein abundance at elution volume of 13 ml, suggesting 147 that the population of purified CFTR molecules is heterogeneous with respect to specific enzyme 148 activity.

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Previous studies found that amphipols do not compete with lipids, which remain associated to  (Figure 3d). 169 We cannot exclude the presence of additional lipids, given the resolution of the solvent system 170 employed for TLC. In fact, we expect that phosphatidylserine (PS) is also associated given its We compared the specific ATPase activities of CFTR purified using amphipol or LMNG after 177 PKA phosphorylation (P) to ensure that both preparations were maximally phosphorylated, a 178 modification known to be important for CFTR function as an enzyme and a channel (Chang,179 Szellas and Nagel 2003). We found that the Km values of ATP were similar for the two 181 samples: Km of 0.27 ± 0.08 mM ATP for the purified P-CFTR in amphipol and Km of 0.32 ± 182 0.14 mM ATP for the purified P-CFTR in LMNG detergent micelles ( Figure 4a). Interestingly, 183 the maximal ATPase activities, after normalization to protein amounts (Refer to CFTR 184 quantification section in Materials and Methods for more details on the method), were 185 significantly higher in the purified P-CFTR: amphipol complexes than in the purified P-CFTR: 186 LMNG micelles (Vmax of 23.90 ± 1.91 nmol phosphate/mg protein/min versus Vmax of 5.54 ± 187 0.66 nmol phosphate/mg protein/min respectively, Figure 4a).

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In order to determine the role of lipids in enhancing the ATPase activity of the amphipol    Thus, we reasoned that CFTR extracted with its associated lipids using amphipol would exhibit a 239 greater response to VX-770 than CFTR extracted with detergents. Now, we show in paired 240 studies, that the fold increase in iodide electrodiffusion caused by VX-770 (1 μM) from 241 proteoliposomes containing amphipol extracted CFTR was approximately twice that measured 242 for proteoliposomes containing LMNG extracted protein (Figure 5c). We interpret these findings 243 to suggest that the lipids present in the CFTR: amphipol complexes facilitate VX-770 binding 244 and/or the conformational changes required for its potentiation. We provide direct evidence that lipid interaction with purified CFTR enhances its catalytic 250 function, an activity that is coupled to its ion channel activity. Furthermore, the lipids associated

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We also report several methodological advances in the study of CFTR. Our studies suggest that 256 purification of membrane proteins that are relatively intractable, such as CFTR, can be achieved

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This is not the first study to show that CFTR purified in detergents can be functionally MD simulations carried out on phosphorylated, ATP-bound hCFTR revealed potential 282 phospholipid binding sites. Importantly, this model was built based on a template structure 283 (zCFTR), which shares 55% of sequence identity with hCFTR and shows a nonconductive state.

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As previously mentioned, preferential binding of POPC was found at the external cleft between 285 TM7 and TM9 and preferential binding of POPS was found at the inward facing gaps between

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We showed previously that the channel activity of detergent purified CFTR was potentiated by  . All systems were NpT-equilibrated in three 10 ns-stages before production runs, 385 successively with protein heavy atoms, protein backbone atoms, and protein Cα atoms restrained.

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All positional restraints used a force constant of 1000 kJ/mol/nm 2 . Random initial velocities were 387 generated for twenty replicas of POPC-embedded system (embedded using either InflateGRO or 388 InflateGRO2 procedures), followed by 1 µs production runs of each replica, for an aggregate 389 total sampling time of 20 µs for POPC-embedded hCFTR. Random initial velocities were 390 generated for ten replicas of the POPS-embedded system (embedded using CHARMM-GUI), 391 followed by 1 µs production runs of each replica, for an aggregate total sampling time of 10 µs 392 for POPS-embedded hCFTR.

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To obtain values of bulk lipid densities for normalizing lipid spatial distribution functions of  The "All Without C-terminus" subset is the "all" subset minus residues 1438-1485 in zCFTR or       highlight regions where differences can be observed between these spatial distribution functions.

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Preferential binding of POPC was found at the external cleft between TM7 and TM9 (region II).

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Preferential binding of POPS was found at the internal clefts between TM10 and TM11 (region 881 I) and between TM4 and TM6 (region III).