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Horizontal Tabs
PDB ID |
Structure Details |
Structure of human KCC1 in complex with ATP (reference map/model) |
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Structure of human KCC1 in complex with ATP (subclass 1) |
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Structure of human KCC1 in complex with ATP (subclass 2) |
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Structure of human KCC3b-PM in NaCl |
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Structure of human KCC3b-PM in NaCl (subclass) |
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Structure of human KCC3b-PM in KCl |
Molecular biology, virus production and protein expression
Full-length and Δ19-deleted human KCC1 (isoform A) and KCC3 isoform B were cloned from the mammalian gene collection (MGC: 1455 and 161519, IMAGE ID: 3349710 and 8991957, respectively) into LIC-adapted pHTBV C-terminally tagged twin-strep, 10-His vector with and without GFP. Full-length NKCC1, KCC2, and phospho-mimetic (S45D, T940D, T997D) and phospho-knockout (S45A, T940A, T997A) constructs of KCC3b were synthesised (GenScript, Twist Bioscience) and subcloned into LIC-adapted pHTBV C-terminally tagged twin-strep, 10-His vector with GFP.
Below are the DNA sequences of the constructs used in this study.
KCC1-Δ19 |
MPHFTVVPVDGPRRGDYDNLEGLSWVDYGERAELDDSDGHGNHRESSPFLSPLEASRGIDYYDRNLALFEEELDIRPKVSSLLGKLVSYTNLTQGAKEHEEAESGEGTRRRAAEAPSMGTLMGVYLPCLQNIFGVILFLRLTWMVGTAGVLQALLIVLICCCCTLLTAISMSAIATNGVVPAGGSYFMISRSLGPEFGGAVGLCFYLGTTFAAAMYILGAIEILLTYIAPPAAIFYPSGAHDTSNATLNNMRVYGTIFLTFMTLVVFVGVKYVNKFASLFLACVIISILSIYAGGIKSIFDPPVFPVCMLGNRTLSRDQFDICAKTAVVDNETVATQLWSFFCHSPNLTTDSCDPYFMLNNVTEIPGIPGAAAGVLQENLWSAYLEKGDIVEKHGLPSADAPSLKESLPLYVVADIATSFTVLVGIFFPSVTGIMAGSNRSGDLRDAQKSIPVGTILAIITTSLVYFSSVVLFGACIEGVVLRDKYGDGVSRNLVVGTLAWPSPWVIVIGSFFSTCGAGLQSLTGAPRLLQAIAKDNIIPFLRVFGHGKVNGEPTWALLLTALIAELGILIASLDMVAPILSMFFLMCYLFVNLACAVQTLLRTPNWRPRFKYYHWALSFLGMSLCLALMFVSSWYYALVAMLIAGMIYKYIEYQGAEKEWGDGIRGLSLSAARYALLRLEEGPPHTKNWRPQLLVLLKLDEDLHVKYPRLLTFASQLKAGKGLTIVGSVIQGSFLESYGEAQAAEQTIKNMMEIEKVKGFCQVVVASKVREGLAHLIQSCGLGGMRHNSVVLGWPYGWRQSEDPRAWKTFIDTVRCTTAAHLALLVPKNIAFYPSNHERYLEGHIDVWWIVHDGGMLMLLPFLLRQHKVWRKCRMRIFTVAQMDDNSIQMKKDLAVFLYHLRLEAEVEVVEMHNSDISAYTYERTLMMEQRSQMLRQMRLTKTEREREAQLVKDRHSALRLESLYSDEEDESAVGADKIQMTWTRDKYMTETWDPSHAPDNFRELVHIKPDQSNVRRMHTAVKLNEVIVTRSHDARLVLLNMPGPPRNSEGDENYMEFLEVLTEGLERVLLVRGGGREV |
KCC3b-WT |
MPHFTVTKVEDPEEGAAASISQEPSLADIKARIQDSDEPDLSQNSITGEHSQLLDDGHKKARNAYLNNSNYEEGDEYFDKNLALFEEEMDTRPKVSSLLNRMANYTNLTQGAKEHEEAENITEGKKKPTKTPQMGTFMGVYLPCLQNIFGVILFLRLTWVVGTAGVLQAFAIVLICCCCTMLTAISMSAIATNGVVPAGGSYFMISRALGPEFGGAVGLCFYLGTTFAAAMYILGAIEIFLVYIVPRAAIFHSDDALKESAAMLNNMRVYGTAFLVLMVLVVFIGVRYVNKFASLFLACVIVSILAIYAGAIKSSFAPPHFPVCMLGNRTLSSRHIDVCSKTKEINNMTVPSKLWGFFCNSSQFFNATCDEYFVHNNVTSIQGIPGLASGIITENLWSNYLPKGEIIEKPSAKSSDVLGSLNHEYVLVDITTSFTLLVGIFFPSVTGIMAGSNRSGDLKDAQKSIPIGTILAILTTSFVYLSNVVLFGACIEGVVLRDKFGDAVKGNLVVGTLSWPSPWVIVIGSFFSTCGAGLQSLTGAPRLLQAIAKDNIIPFLRVFGHSKANGEPTWALLLTAAIAELGILIASLDLVAPILSMFFLMCYLFVNLACALQTLLRTPNWRPRFRYYHWALSFMGMSICLALMFISSWYYAIVAMVIAGMIYKYIEYQGAEKEWGDGIRGLSLSAARFALLRLEEGPPHTKNWRPQLLVLLKLDEDLHVKHPRLLTFASQLKAGKGLTIVGSVIVGNFLENYGEALAAEQTIKHLMEAEKVKGFCQLVVAAKLREGISHLIQSCGLGGMKHNTVVMGWPNGWRQSEDARAWKTFIGTVRVTTAAHLALLVAKNISFFPSNVEQFSEGNIDVWWIVHDGGMLMLLPFLLKQHKVWRKCSIRIFTVAQLEDNSIQMKKDLATFLYHLRIEAEVEVVEMHDSDISAYTYERTLMMEQRSQMLRHMRLSKTERDREAQLVKDRNSMLRLTSIGSDEDEETETYQEKVHMTWTKDKYMASRGQKAKSMEGFQDLLNMRPDQSNVRRMHTAVKLNEVIVNKSHEAKLVLLNMPGPPRNPEGDENYMEFLEVLTEGLERVLLVRGGGSEVITIYS |
KCC3b-PM |
MPHFTVTKVEDPEEGAAASISQEPSLADIKARIQDSDEPDLSQNDITGEHSQLLDDGHKKARNAYLNNSNYEEGDEYFDKNLALFEEEMDTRPKVSSLLNRMANYTNLTQGAKEHEEAENITEGKKKPTKTPQMGTFMGVYLPCLQNIFGVILFLRLTWVVGTAGVLQAFAIVLICCCCTMLTAISMSAIATNGVVPAGGSYFMISRALGPEFGGAVGLCFYLGTTFAAAMYILGAIEIFLVYIVPRAAIFHSDDALKESAAMLNNMRVYGTAFLVLMVLVVFIGVRYVNKFASLFLACVIVSILAIYAGAIKSSFAPPHFPVCMLGNRTLSSRHIDVCSKTKEINNMTVPSKLWGFFCNSSQFFNATCDEYFVHNNVTSIQGIPGLASGIITENLWSNYLPKGEIIEKPSAKSSDVLGSLNHEYVLVDITTSFTLLVGIFFPSVTGIMAGSNRSGDLKDAQKSIPIGTILAILTTSFVYLSNVVLFGACIEGVVLRDKFGDAVKGNLVVGTLSWPSPWVIVIGSFFSTCGAGLQSLTGAPRLLQAIAKDNIIPFLRVFGHSKANGEPTWALLLTAAIAELGILIASLDLVAPILSMFFLMCYLFVNLACALQTLLRTPNWRPRFRYYHWALSFMGMSICLALMFISSWYYAIVAMVIAGMIYKYIEYQGAEKEWGDGIRGLSLSAARFALLRLEEGPPHTKNWRPQLLVLLKLDEDLHVKHPRLLTFASQLKAGKGLTIVGSVIVGNFLENYGEALAAEQTIKHLMEAEKVKGFCQLVVAAKLREGISHLIQSCGLGGMKHNTVVMGWPNGWRQSEDARAWKTFIGTVRVTTAAHLALLVAKNISFFPSNVEQFSEGNIDVWWIVHDGGMLMLLPFLLKQHKVWRKCSIRIFTVAQLEDNSIQMKKDLATFLYHLRIEAEVEVVEMHDSDISAYTYERDLMMEQRSQMLRHMRLSKTERDREAQLVKDRNSMLRLTSIGSDEDEETETYQEKVHMDWTKDKYMASRGQKAKSMEGFQDLLNMRPDQSNVRRMHTAVKLNEVIVNKSHEAKLVLLNMPGPPRNPEGDENYMEFLEVLTEGLERVLLVRGGGSEVITIYS |
KCC3b-PKO |
MPHFTVTKVEDPEEGAAASISQEPSLADIKARIQDSDEPDLSQNAITGEHSQLLDDGHKKARNAYLNNSNYEEGDEYFDKNLALFEEEMDTRPKVSSLLNRMANYTNLTQGAKEHEEAENITEGKKKPTKTPQMGTFMGVYLPCLQNIFGVILFLRLTWVVGTAGVLQAFAIVLICCCCTMLTAISMSAIATNGVVPAGGSYFMISRALGPEFGGAVGLCFYLGTTFAAAMYILGAIEIFLVYIVPRAAIFHSDDALKESAAMLNNMRVYGTAFLVLMVLVVFIGVRYVNKFASLFLACVIVSILAIYAGAIKSSFAPPHFPVCMLGNRTLSSRHIDVCSKTKEINNMTVPSKLWGFFCNSSQFFNATCDEYFVHNNVTSIQGIPGLASGIITENLWSNYLPKGEIIEKPSAKSSDVLGSLNHEYVLVDITTSFTLLVGIFFPSVTGIMAGSNRSGDLKDAQKSIPIGTILAILTTSFVYLSNVVLFGACIEGVVLRDKFGDAVKGNLVVGTLSWPSPWVIVIGSFFSTCGAGLQSLTGAPRLLQAIAKDNIIPFLRVFGHSKANGEPTWALLLTAAIAELGILIASLDLVAPILSMFFLMCYLFVNLACALQTLLRTPNWRPRFRYYHWALSFMGMSICLALMFISSWYYAIVAMVIAGMIYKYIEYQGAEKEWGDGIRGLSLSAARFALLRLEEGPPHTKNWRPQLLVLLKLDEDLHVKHPRLLTFASQLKAGKGLTIVGSVIVGNFLENYGEALAAEQTIKHLMEAEKVKGFCQLVVAAKLREGISHLIQSCGLGGMKHNTVVMGWPNGWRQSEDARAWKTFIGTVRVTTAAHLALLVAKNISFFPSNVEQFSEGNIDVWWIVHDGGMLMLLPFLLKQHKVWRKCSIRIFTVAQLEDNSIQMKKDLATFLYHLRIEAEVEVVEMHDSDISAYTYERALMMEQRSQMLRHMRLSKTERDREAQLVKDRNSMLRLTSIGSDEDEETETYQEKVHMAWTKDKYMASRGQKAKSMEGFQDLLNMRPDQSNVRRMHTAVKLNEVIVNKSHEAKLVLLNMPGPPRNPEGDENYMEFLEVLTEGLERVLLVRGGGSEVITIYS |
NKCC1 |
MEPRPTAPSSGAPGLAGVGETPSAAALAAARVELPGTAVPSVPEDAAPASRDGGGVRDEGPAAAGDGLGRPLGPTPSQSRFQVDLVSENAGRAAAAAAAAAAAAAAAGAGAGAKQTPADGEASGESEPAKGSEEAKGRFRVNFVDPAASSSAEDSLSDAAGVGVDGPNVSFQNGGDTVLSEGSSLHSGGGGGSGHHQHYYYDTHTNTYYLRTFGHNTMDAVPRIDHYRHTAAQLGEKLLRPSLAELHDELEKEPFEDGFANGEESTPTRDAVVTYTAESKGVVKFGWIKGVLVRCMLNIWGVMLFIRLSWIVGQAGIGLSVLVIMMATVVTTITGLSTSAIATNGFVRGGGAYYLISRSLGPEFGGAIGLIFAFANAVAVAMYVVGFAETVVELLKEHSILMIDEINDIRIIGAITVVILLGISVAGMEWEAKAQIVLLVILLLAIGDFVIGTFIPLESKKPKGFFGYKSEIFNENFGPDFREEETFFSVFAIFFPAATGILAGANISGDLADPQSAIPKGTLLAILITTLVYVGIAVSVGSCVVRDATGNVNDTIVTELTNCTSAACKLNFDFSSCESSPCSYGLMNNFQVMSMVSGFTPLISAGIFSATLSSALASLVSAPKIFQALCKDNIYPAFQMFAKGYGKNNEPLRGYILTFLIALGFILIAELNVIAPIISNFFLASYALINFSVFHASLAKSPGWRPAFKYYNMWISLLGAILCCIVMFVINWWAALLTYVIVLGLYIYVTYKKPDVNWGSSTQALTYLNALQHSIRLSGVEDHVKNFRPQCLVMTGAPNSRPALLHLVHDFTKNVGLMICGHVHMGPRRQAMKEMSIDQAKYQRWLIKNKMKAFYAPVHADDLREGAQYLMQAAGLGRMKPNTLVLGFKKDWLQADMRDVDMYINLFHDAFDIQYGVVVIRLKEGLDISHLQGQEELLSSQEKSPGTKDVVVSVEYSKKSDLDTSKPLSEKPITHKVEEEDGKTATQPLLKKESKGPIVPLNVADQKLLEASTQFQKKQGKNTIDVWWLFDDGGLTLLIPYLLTTKKKWKDCKIRVFIGGKINRIDHDRRAMATLLSKFRIDFSDIMVLGDINTKPKKENIIAFEEIIEPYRLHEDDKEQDIADKMKEDEPWRITDNELELYKTKTYRQIRLNELLKEHSSTANIIVMSLPVARKGAVSSALYMAWLEALSKDLPPILLVRGNHQSVLTFYS |
Baculoviral DNA from the transformation of DH10Bac were used to transfect Sf9 cells to produce baculovirus particles for transduction. Virus was amplified by transducing mid-log Sf9 cells (2 × 106 cells mL−1) grown in Sf-900™ II media supplemented with 2 % fetal bovine serum (Thermo Fisher Scientific). Cells were incubated on an orbital shaker for 65 h at 27 °C in 1 L shaker flasks. Baculovirus were harvested by centrifugation at 900g for 10 min with the virus contained in the supernatant.
1 L of Expi293F™ GnTI- cell cultures (2 × 106 cells mL−1) in Freestyle 293™ Expression Medium (Thermo Fisher Scientific) were infected with high-titre P3 baculovirus (3 % v/v) in the presence of 5 mM sodium butyrate in a 2 L roller bottle (Biofil). Cells were grown in a humidity-controlled orbital shaker for 48 h at 37 °C with 8 % CO2 before being harvested by centrifugation at 900g for 10 min, washed with phosphate-buffered saline, and pelleted again prior to flash freezing in liquid nitrogen (LN2), then stored at −80 °C until needed.
Protein purification of digitonin samples
Whole cell pellets expressing varying constructs of NKCC1, KCC1, KCC2 and KCC3b were resuspended to a total volume of 50 mL per 15 g of cell pellet with buffer A (150 mM NaCl, 20 mM HEPES pH 7.5) supplemented with, 0.7 % w/v Lauryl Maltose Neopentyl Glycol, LMNG (Generon), and 0.07 % cholesteryl hemisuccinate, CHS (Generon). The cells were solubilised at 4 °C for 1 h with gentle rotation. Cell debris was pelleted at 50,000g for 30 min. The clarified lysate was added to 0.5 mL bed volume of Strep-Tactin SuperFlow (IBA) per 50 mL of lysate, and allowed to bind at 4 °C for 1 h. The resin was collected on a gravity-flow column and washed with buffer B (buffer A with 0.003 % w/v LMNG and 0.0003 % w/v CHS), and then with buffer B supplemented with 1 mM ATP and 5 mM MgCl2. Protein was eluted with 7 CV of buffer B supplemented with 5 mM D-desthiobiotin followed by tag-cleavage by TEV protease overnight and reverse purification. For LMNG/CHS condition, the samples were subjected to size exclusion chromatography pre-equilibrated with Buffer B. For digitonin condition, buffer A supplemented with 0.04 % digitonin (Apollo Scientific) was used for equilibration instead. Peak fractions were pooled and concentrated to 5 µM for LMNG/CHS sample and 50 µM for digitonin sample for subsequent experiments.
Nanodisc sample was prepared similarly to the detergent samples with a few exceptions. After washing the protein-bound Strep-Tactin resin, buffer A supplemented with 0.5 % LMNG, 0.05% CHS and 0.125 % soy azolectin (Sigma) was added to a final LMNG concentration of 0.2 %, and purified MSP E3D1 protein to a final concentration of 0.5 mg/mL. The slurry was incubated on a rotating wheel for 15 min, then 100 mg of washed Biobead SM-2 per mL resin was added, followed by further incubation for a minimum of 4 hours. Subsequent purifications were then performed with buffer A.
Cryo-electron microscopy sample preparation, data collection and data processing
All samples were frozen on Quantifoil Au R1.2/1.3 mesh 300 grids freshly glow discharged for 30 s, with plunge freezing performed on Vitrobot Mark IV (Thermo Fisher Scientific) chamber set to 80-100 % humidity and 4 °C. For LMNG/CHS and MSP E3D1 conditions, blotting time was set to 1.0 – 1.5 s, and for digitonin conditions it was set to 3.5 – 5.0 s after 30 s wait time.
The cryo-EM datasets were collected on Titan Krioses (Thermo Fisher Scientific) operating at 300 keV. Super-resolution dose-fractionated microcrographs (0.3255 Å pixel-1 or 0.415 Å pixel-1) were collected on a K3 (Gatan) detector by image shift collection two exposures per hole with a total dose of 40 – 45 e- Å-2. Micrographs were binned 2 x 2 during motion correction with 5 by 5 patches using MotionCor2. Motion-corrected images were then processed in Cryosparc 2.11.0 where defocus values were determined by Patch CTF function on Cryosparc. Particles were picked with blob picker function, and extracted particles were subjected to two cycles of 2D classification. Particles from good classes were used to generate three ab-initio models. Particles in the good model class were then used for non-uniform refinement function with C2 symmetry using the ab-initio model as reference.
Rb+ flux assays
Defolliculated stage V–VI Xenopus laevis oocytes were microinjected with KCC-derived cRNAs (same amount between WT and mutants for each of the KCC1 and KCC3b isoforms) and maintained in Barth medium for 3 d at 18 °C in the presence of 1.5 mM furosemide. Water-injected oocytes were used as controls. Before the transport assay, furosemide was removed through several washes in plain Barth medium. Carrier activity was assessed at room temperature through Rb+ influx assays under isotonic and hypotonic conditions. The experiments were carried out more specifically by incubating oocytes for 1 h in a hypotonic solution (125 mOsM) or in an isotonic solution (200 mOsM) and reincubating them afterwards for 45 min in an isotonic salt-added physiological solution (7 mM Rb+, 86 mM Cl–) in the presence or absence of 1.5 mM furosemide. At the end of flux assays, oocytes were washed several times in a refrigerated Rb+-free solution, lysed in pure nitric acid and assayed for Rb+ content (1 oocyte/sample) by atomic absorption spectrophotometry (Varian AA240). Transport data for oocytes are expressed in this work as mean (± S.E.) background-subtracted transport rates in 10 oocytes among 3 to 6 experiments.
LC-MS analysis for small molecule identification
The purified KCC1 protein sample was initially buffer exchange into 200 mM ammonium acetate at pH 7.4 using a bipspin column (Bio-rad) and resuspended in 100 μL of water with 0.1 % formic acid to be analyzed by LC-MS/MS. The separation was performed using an Ascentis Express C18 analytical column (0.3 x 150 mm, 2.7 μm) at 15 μL.min-1 using the isocratic elution with following mobile phases, A – water with 0.1 % formic acid and B – acetonitrile with 0.1 % formic acid at 97:3 (v/v), respectively. The sample volume injected was 1 μL and the total run time was 3 min. All measurements were performed using the Orbitrap Eclipse Tribrid mass spectrometer coupled with Ultimate 3000 binary pump. The MS was operated in negative polarity and the ionization conditions were 275 °C for capillary temperature (ion transfer tube), 20 °C for vaporizer temperature and 3500 V for spray voltage. Thermo Xcalibur software was used for data processing.
NanoDSF measurements
All KCC variants were diluted up to 0.2 mg/ml in protein buffer (20 mM HEPES pH 7.4, 150 mM NaCl, digitonin). 10 x nucleotide stock solutions in were prepared in protein buffer. Protein and nucleotides were mixed and incubated on ice for 30 min. NT.Plex nanoDSF Grade High Sensitivity Capillaries (NanoTemper) were filled with 10-µl protein sample. Melting curves were determined in triplicates using Prometheus NT.48 by monitoring the intrinsic protein fluorescence signal as a measure of its folding state during a temperature ramp (1 °C/min increase) from 20 to 95°C. Exemplary melting curves are shown in Supplementary Figure 3. The melting temperature was determined by averaging the melting temperature of the triplicate measurements.
Molecular Dynamics Simulations
The MD simulations were carried out using Desmond simulation package of Schrödinger LLC. The NPT ensemble with the temperature 300 K and a pressure 1 bar was applied in all runs. The simulation length was 500 ns with a relaxation time 1 ps for the ligand ATP. The OPLS3e force field parameters were used in all simulations. The long-range electrostatic interactions were calculated using the particle mesh Ewald method. The cutoff radius in Coulomb interactions was 9.0 Å. The water molecules were explicitly described using the simple point charge model. The Martyna–Tuckerman–Klein chain coupling scheme with a coupling constant of 2.0 ps was used for the pressure control and the Nosé–Hoover chain coupling scheme for the temperature control. Non-bonded forces were calculated using an r-RESPA integrator where the short-range forces were updated every step and the long-range forces were updated every three steps. The trajectories were saved at 200 ps intervals for analysis. The behaviour and interactions between the ligands and protein were analysed using the Simulation Interaction Diagram tool implemented in Desmond MD package. The stability of MD simulations was monitored by looking on the rmsd of the ligand and protein atom positions in time.
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