Manuscript submitted to: Volume 2, Issue 2, 131-152. AIMS Biophysics Received date 16 February 2015, Accepted date 04 May 2015, Published date 13 May 2015 DOI: 10.3934/biophy.2015.2.131 Research article Structure of the cystic fibrosis transmembrane conductance regulator in the inward-facing conformation revealed by single particle electron microscopy Ateeq Al-Zahrani 1,2, Natasha Cant 1, Vassilis Kargas 1,3, Tracy Rimington 1, Luba Aleksandrov 4, John R. Riordan 4, and Robert C. Ford 1, * 1 2 3 4 Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, UK University College in Qunfudah, The University of Umm-Alqura, Kingdom of Saudi Arabia Bioinformatics Institute, 30 Biopolis Street, #07-01 Matrix, 138671 Singapore Department of Biochemsitry and Biophysics, University of North Carolina, Chapel Hill, 6107 Thurston-Bowles, Campus Box 7248, Chapel Hill, NC 27599, USA * Correspondence: Email: robert.ford@manchester.ac.uk. Supplementary information
Supplementary Figure 1 Chicken-CFTR Purification by Ni-NTA chromatography in the detergent DDM. 10% SDS-PAGE. 10ul sample loaded per well: Left image: Detection using the GFP tag. Right image: Detection using Coomassie stain. 1 2 3 4 5 6 7 8 9 250 130 100 70 55 250 130 100 70 55 CFTR RPL3 Elution fractions 2-4 at 21, 25, 80µg/ml, respectively were the purest. Each fraction was 2ml, yielding about 0.25mg CFTR in these fractions. Elution was in buffer: 50mMTris ph8, 20% glycerol, 1M NaCl, 400mM Imidazole, 1mM DTT, 0.1% DDM. Elution fractions emerging last at 400mM imidazole (4-6) are enriched in the yeast ribosomal subunit RPL3.
Supplementary Figure 2 Chicken-CFTR Purification by Ni-NTA chromatography in the detergent LPG. 250-130 - 100-70 - 55 - CFTR 35 -
Supplementary Figure 3: Left panel: CFTR expressed in yeast and purified in DDM was treated with no enzyme (-) or protein kinase A and ATP (PKA) or alkaline phosphatase (CIP) at 4 C overnight and analysed by SDS-PAGE. CFTR was detected by the GFPfluorescence (left) and phosphorylated CFTR is shown by Pro-Q Diamond staining (right). CFTR appears to be similarly phosphorylated before and after PKA treatment and this phosphorylation can be removed by protein phosphatase (CIP). The migration of the phosphorylated CFTR band is not observed to be slower than the dephosphorylated protein, unlike for mammalian cell expression of CFTR.
Supplementary Figure 4: (A) CFTR C-terminal peptide and NHERF1-PDZ1 when co-expressed in Escherichia coli can be purified as a complex: Non-denaturing SDS-PAGE of the co-expressed C-terminus and NHERF1 PDZ 1 complex. Lanes: 1- IMAC-Unbound material (after thrombin cleavage of the His-tag), 2- Retentate of a 50 kda cut-off concentrator, 3- Filtrate of the concentrator, 4- Final concentrated complex showing the two components. (B) Purification of human NHERF1 PDZ1 (amino acid residues 11-99) was expressed alone as a glutathione S-transferase fusion protein in Escherichia coli, affinity-purified and then cleaved from the GST tag with thrombin. Lanes: 1. Molecular mass markers. 2. IMAC-Unbound material (after thrombin cleavage). 3. Retentate of a 50 kda concentrator. 4. Filtrate of 50 kda concentrator. 5. Retentate of 3 kda concentrator. Composition of the various bands was checked by mass spectrometry (Supplementary figure 5). A 1 2 3 4 B complex PDZ1 C-term 1 2 3 4 5
Supplementary Figure 5: Co- expression and co- purifica:on of untagged NHERF1 PDZ1 and tagged CFTR C- terminal pep:de: Mass spectrometry data confirming the iden:ty of the individual components and the complex. NHERF1-PDZ1 NHERF1-PDZ1 + C-term (Complex)??????????????? NHERF1 Cterm C-terminal peptide
Supplementary Figure 6: Size- exclusion chromatography/mals of the expressed and purified NHERF1- PDZ1 confirms the homodimeric organisa:on of the 10kD protein that was observed previously in X- ray crystallography studies. 20kDa
Supplementary Figure 7: Image processing of nega:vely- stained dimeric CFTR par:cles (A) A field of dimeric human CFTR par:cles purified in DDM in the presence of purified NHERF1 PDZ1. Circled par:cles represent those selected for processing and meet the criteria described in the Methods sec:on. Small clusters of touching par:cles and aggregates (arrows) were not selected. Some significantly smaller par:cles are also observable (squares), which may represent clusters of dimeric NHERF1 PDZ1 domains (see text). The scale bar represents 20 nm. Lower panel shows projec:on class averages of the CFTR par:cles according to their orienta:on on the grid support surface. (B) Projec:ons of the final 3D structure (even numbers) and the corresponding average of the aligned par:cles corresponding to that projec:on (odd numbers). (C) Resolu:on es:ma:on of the CFTR structures using the Fourier Shell Correla:on between two sub- sets of each data set. Solid line: CFTR (control dataset). Dashed line: CFTR+NHERF1 PDZ1.
Supplementary Figure 8 Alignment of part of TMD2 of CFTR (ABCC7) with other ABCC family members. Transmembrane spans 10,11 and 12 are highlighted in orange. Highlighted in yellow are ICL regions equivalent to those forming a glutathione binding site in the Atm1 transporter structure (PDBID=2MYH). The Atm1 sequence at these positions is given immediately above the yellow highlighted regions, and residues forming interactions with the ligand are coloured red. TM9 TM10 RTHFR ABCC7 999 L-IVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTS-QQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFET 1076 ABCC4 848 L-QVVGVVSVAVAVIPWIAIPLVPLGIIFIFLRRYFLETS-RDVKRLESTTRSPVFSHLSSSLQGLWTIRAYKAEERCQE 925 ABCC5 998 IlVFFCVGMIAGVFPWFLVAVGPLVIL--FSVLHIVSRVLiRELKRLDNITQSPFLSHITSSIQGLATIHAYNKGQEFLH 1075 ABCC2 1107 L-GIISTLVMICMATPVFTIIVIPLGIIYVSVQMFYVSTS-RQLRRLDSVTRSPIYSHFSETVSGLPVIRAFEHQQRFLK 1184 ABCC11 946 L-MVIAVLLIVSVLSPYILLMGAIIMVICFIYYMMFKKAI-GVFKRLENYSRSPLFSHILNSLQGLSSIHVYGKTEDFIS 1023 ABCC12 935 F-MVVFILVILAAVFPAVLLVVASLAVGFFILLRIFHRGV-QELKKVENVSRSPWFTHITSSMQGLGIIHAYGKKESCIT 1012 ABCC3 1095 F-NAISTLVVIMASTPLFTVVILPLAVLYTLVQRFYAATS-RQLKRLESVSRSPIYSHFSETVTGASVIRAYNRSRDFEI 1172 ABCC1 1099 F-NVIGACIVILLATPIAAIIIPPLGLIYFFVQRFYVASS-RQLKRLESVSRSPVYSHFNETLLGVSVIRAFEEQERFIH 1176 ABCC9 1119 L-LCLSAIGMISYATPVFLVALLPLGVAFYFIQKYFRVAS-KDLQELDDSTQLPLLCHFSETAEGLTTIRAFRHETRFKQ 1196 ABCC8 1147 L-LCVSALAVISYVTPVFLVALLPLAIVCYFIQKYFRVAS-RDLQQLDDTTQLPLLSHFAETVEGLTTIRAFRYEARFQQ 1224 ABCC6 1071 F-GLLEVSLVVAVATPLATVAILPLFLLYAGFQSLYVVSS-CQLRRLESASYSSVCSHMAETFQGSTVVRAFRTQAPFVA 1148 ABCC10 1051 A-GLLGLLAVLGSGLPWLLLLLPPLSIMYYHVQRHYRASS-RELRRLGSLTLSPLYSHLADTLAGLSVLRATGATYRFEE 1128 TM11 TM12 LNSGQNLI LNFLGSVYRDL ABCC7 1077 LFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGE--GEGRV-GIILTLAMNIMSTLQWAVNSSIDV 1153 ABCC4 926 LFDAHQDLHSEAWFLFLTTSRWFAVRLDAICAMFVIIVAFGSLILAKT--LDAGQVGLALSYALTLMGMFQWCVRQSAEV 1003 ABCC5 1076 RYQELLDDNQAPFFLFTCAMRWLAVRLDLISIALITTTGLMIVLMHGQ--IPPAYAGLAISYAVQLTGLFQFTVRLASET 1153 ABCC2 1185 HNEVRIDTNQKCVFSWITSNRWLAIRLELVGNLTVFFSALMMVIYRDT--LSGDTVGFVLSNALNITQTLNWLVRMTSEI 1262 ABCC11 1024 QFKRLTDAQNNYLLLFLSSTRWMALRLEIMTNLVTLAVALFVAFGISS--TPYSFKVMAVNIVLQLASSFQATARIGLET 1101 ABCC12 1013 Y----------HLLYFNCALRWFALRMDVLMNILTFTVALLVTLSFSS--ISTSSKGLSLSYIIQLSGLLQVCVRTGTET 1080 ABCC3 1173 ISDTKVDANQRSCYPYIISNRWLSIGVEFVGNCVVLFAALFAVIGRSS--LNPGLVGLSVSYSLQVTFALNWMIRMMSDL 1250 ABCC1 1177 QSDLKVDENQKAYYPSIVANRWLAVRLECVGNCIVLFAALFAVISRHS--LSAGLVGLSVSYSLQVTTYLNWLVRMSSEM 1254 ABCC9 1197 RMLELTDTNNIAYLFLSAANRWLEVRTDYLGACIVLTASIASISGSS----NSGLVGLGLLYALTITNYLNWVVRNLADL 1272 ABCC8 1225 KLLEYTDSNNIASLFLTAANRWLEVRMEYIGACVVLIAAVTSISNSLHreLSAGLVGLGLTYALMVSNYLNWMVRNLADM 1304 ABCC6 1149 QNNARVDESQRISFPRLVADRWLAANVELLGNGLVFAAATCAVLSKAH--LSAGLVGFSVSAALQVTQTLQWVVRNWTDL 1226 ABCC10 1129 ENLRLLELNQRCQFATSATMQWLDIRLQLMGAAVVSAIAGIALVQHQQglANPGLVGLSLSYALSLTGLLSGLVSSFTQT 1208
Supplementary Figure 9 Alignment of part of TMD1 of CFTR (ABCC7) with other ABCC family members. Transmembrane spans 4,5 and 6 are highlighted in orange. Highlighted in yellow are regions where a glutathione binding site exists in the Atm1 transporter structure (PDBID=2MYH). The Atm1 sequence at these positions is given immediately above the yellow highlighted regions, and residues forming interactions with the ligand are coloured red. TM4 TM5 RTHFR LNSGQNLI ABCC7 230 LIVLALFQAGLG-RMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSS 308 ABCC4 244 LIILLPLQSCFG-KLFSSLRSKTATFTDARIRTMNEVITGIRIIKMYAWEKSFSNLITNLRKKEISKILRSSCLRGMN-L 321 ABCC5 326 FILFYPAMM-FAsRLTAYFRRKCVAATDERVQKMNEVLTYIKFIKMYAWVKAFSQSVQKIREEERRILEKAGYFQSITVG 404 ABCC2 470 MVLVIPINAILS-TKSKTIQVKNMKNKDKRLKIMNEILSGIKILKYFAWEPSFRDQVQNLRKKELKNLLAFSQLQCVVIF 548 ABCC11 310 YLLVFPLAV-FMtRMAVKAQHHTSEVSDQRIRVTSEVLTCIKLIKMYTWEKPFAKIIEDLRRKERKLLEKCGLVQSLTSI 388 ABCC12 270 YVIFIPVQM-FMaKLNSAFRRSAILVTDKRVQTMNEFLTCIRLIKMYAWEKSFTNTIQDIRRRERKLLEKAGFVQSGNSA 348 ABCC3 459 MVLLIPLNGAVA-VKMRAFQVKQMKLKDSRIKLMSEILNGIKVLKLYAWEPSFLKQVEGIRQGELQLLRTAAYLHTTTTF 537 ABCC1 473 MVLMVPVNAVMA-MKTKTYQVAHMKSKDNRIKLMNEILNGIKVLKLYAWELAFKDKVLAIRQEELKVLKKSAYLSAVGTF 551 ABCC9 463 IVLLAPIQYFIA-TKLAEAQKSTLDYSTERLKKTNEILKGIKLLKLYAWEHIFCKSVEETRMKELSSLKTFALYTSLSIF 541 ABCC8 467 IILLAPVQYFVA-TKLSQAQRSTLEYSNERLKQTNEMLRGIKLLKLYAWENIFRTRVETTRRKEMTSLRAFAIYTSISIF 545 ABCC6 459 FLSLLPLNFFIS-KKRNHHQEEQMRQKDSRARLTSSILRNSKTIKFHGWEGAFLDRVLGIRGQELGALRTSGLLFSV--S 535 ABCC10 430 ALLLVPVNKVIA-TRIMASNQEMLQHKDARVKLVTELLSGIRVIKFCGWEQALGARVEACRARELGRLRVIKYLDAAC-- 506 TM5 TM6 LNFLGSVYRDL ABCC7 309 AFFFSGF-FVV-FLSVLPY--ALIKG-IILRKIFTTISFCIVLRMAVTRqFPWAVQTWYDSLGAINKIQDFLQKQEYKTL 383 ABCC4 322 ASFFSASKIIV-FVTFTTYvL--LGSVITASRVFVAVTLYGAVRLTVTLfFPSAIERVSEAIVSIRRIQTFLLLDEISQR 398 ABCC5 405 VAPIVVV-IAS-VVTFSVH--MTLGFDLTAAQAFTVVTVFNSMTFAL-KvTPFSVKSLSEASVAVDRFKSLFLMEEVHMI 479 ABCC2 549 VFQLTPV-LVS-VVTFSVYvLVDSNNILDAQKAFTSITLFNILRFPLSM-LPMMISSMLQASVSTERLEKYLGGDDLDTS 625 ABCC11 389 TLFIIPT-VAT-AVWVLIH--TSLKLKLTASMAFSMLASLNLLRLSV-FfVPIAVKGLTNSKSAVMRFKKFFLQESPVFY 463 ABCC12 349 LAPIVST-IAI-VLTLSCH--ILLRRKLTAPVAFSVIAMFNVMKFSI-AiLPFSIKAMAEANVSLRRMKKILIDKSPPSY 423 ABCC3 538 TWMCSPFLVTL-ITLWVYV-YVDPNNVLDAEKAFVSVSLFNILRLPLNM-LPQLISNLTQASVSLKRIQQFLSQEELDPQ 614 ABCC1 552 TWVCTPFLVAL-CTFAVYV-TIDENNILDAQTAFVSLALFNILRFPLNI-LPMVISSIVQASVSLKRLRIFLSHEELEPD 628 ABCC9 542 MNAAIPIAAVL-ATFVTH--AYASGNNLKPAEAFASLSLFHILVTPLFL-LSTVVRFAVKAIISVQKLNEFLLSDEIGDD 617 ABCC8 546 MNTAIPIAAVL-ITFVGHV-SFFKEADFSPSVAFASLSLFHILVTPLFL-LSSVVRSTVKALVSVQKLSEFLSSAEIREE 622 ABCC6 536 LVSFQVSTFLV-ALVVFAVhTLVAENAMNAEKAFVTLTVLNILNKAQAF-LPFSIHSLVQARVSFDRLVTFLCLEEVDPG 613 ABCC10 507 VYLWAALPVVIsIVIFITY--VLMGHQLTATKVFTALALVRMLILPLNN-FPWVINGLLEAKVSLDRIQLFLDLPNHNPQ 583