The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A
- Department of Crystallography and Structural Biology, Institute of Physical-Chemistry 'Blas Cabrera', CSIC, Spain
- Center for Biomolecular Structure and Dynamics, and Division of Biological Sciences, University of Montana, United States
- Institute for Biocomputation and Physics of Complex Systems (BIFI) and Laboratorio de Microscopías Avanzadas (LMA), University of Zaragoza, Spain
- Department of Neurobiology, Instituto Ramón y Cajal de Investigación Sanitaria, Hospital Universitario Ramón y Cajal, Spain
- AFFINImeter Scientific & Development team, Software 4 Science Developments, Spain
- Departamento de Física Aplicada, Universidad de Santiago de Compostela, Spain
- Department of Biological Physical-Chemisty, Institute of Physical-Chemistry 'Blas Cabrera', CSIC, Spain
- Ciber of Respiratory Diseases, ISCIII, Spain
- Department of Systems Biology, Universidad de Alcala, Spain
Figures
Figure 1 with 1 supplement
The structure of NCS/target complexes.
(A) Ribbon representation of NCS protein structures bound to their targets. NCS-1/D2R (PDB: 5AER, Pandalaneni et al., 2015), NCS-1/Grk1 (PDB: 5AFP, Pandalaneni et al., 2015), Frq1/Pik1 (PDB: 2JU0, St…
Figure 1—figure supplement 1
Structural comparison of hNCS-1/Ric-8A-P with other NCS-1 complexes.
(A, B) Superposition of NCS-1/Ric-8A-P (only Ric-8A is shown in pink ribbons) with other NCS-1 structures in complex with regulatory ligands, the protein-protein interaction (PPI) inhibitor FD-44 (Ma…
Figure 2
The assembly of rat and human NCS-1/Ric-8A complexes.
(A) Ca2+ dependency of the interaction of the rat complex. Size exclusion chromatograms after assemblies: (i) in Ca2+-free conditions (gray), (ii) with Ca2+-preloaded NCS-1ΔH10 (magenta), and (iii) …
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Figure 2—source data 1
Original gels, WBs and SEC and nano-DSF raw data.
- https://cdn.elifesciences.org/articles/86151/elife-86151-fig2-data1-v2.zip
Figure 3 with 2 supplements
The structure of hNCS-1 bound to a Ric-8A peptide.
(A) Ribbon representation of the hNCS-1ΔH10/Ric-8A-P3 complex. Two views are displayed. The NCS-1 structure is shown in light purple, while Ric-8A-P3 is shown in light pink. The N- and C-termini are …
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Figure 3—source data 1
Original WBs.
- https://cdn.elifesciences.org/articles/86151/elife-86151-fig3-data1-v2.zip
Figure 3—figure supplement 1
Structure resolution of hRic-8A peptides bound to NCS-1.
(A) Structure 1 showing the 2Fo-Fc electron density map (green) of Ric-8A-P2 (stick mode, pink). The molecular surface of NCS-1 is depicted. Squares represent magnifications of R1 and R2 regions. (B)…
Figure 3—figure supplement 2
The hNCS-1/Ric-8A-P protein-protein interface.
(A) Ribbon representation of NCS-1. Helices are labeled and residues implicated in Ric-8A recognition are displayed as light purple sticks. (B) H-bonds (black dashes) between NCS-1 (gray) and Ric-8A …
Figure 4 with 2 supplements
NCS-1 Ca2+ binding sites.
(A) Identification of Ca2+, Mg2+, and Na+ ions in the hNCS-1ΔH10/Ric-8A-P3 complex (Structure 2, see Table 1). Top: Electron density at EF-hands EF-2, -3 and -4. The 2Fo-Fc electron density map …
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Figure 4—source data 1
Raw chromatograms, nano-DSF and Bli data.
- https://cdn.elifesciences.org/articles/86151/elife-86151-fig4-data1-v2.zip
Figure 4—figure supplement 1
Isothermal titration calorimetry.
Thermodynamics of Ca2+ binding to NCS-1 in Na+ (top panels) or K+ (bottom panels) containing buffers. Experimental conditions as in Figure 4D. Raw data (left panels) and binding isotherms (central …
Figure 4—figure supplement 2
Biolayer interferometry (BLI) control experiments.
(A) Nano-differential scanning fluorimetry (nano-DSF) curve of the His-NCS-1 sample used in the BLI assay. (B) Representative BLI sensogram of the binding of His-NCS-1 to the Ni-NTA biosensor and …
Figure 5 with 1 supplement
Ric-8A phosphorylation in the context of the NCS-1/Ric-8A complex.
(A) Co-IP protein-protein interaction assay of hNCS-1 and V5-tagged full-length hRic-8A wild-type (WT) (hRic-8A-WT) and a non-phosphorylatable mutant (Ric-8A-P-Mut; S436A, T441A) in HEK293 cells. (B)…
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Figure 5—source data 1
Original WBs and raw chromatogram data.
- https://cdn.elifesciences.org/articles/86151/elife-86151-fig5-data1-v2.zip
Figure 5—figure supplement 1
Analysis of phosphorylated rRic-8A-452 by mass spectrometry.
MS/MS fragmentation spectra (top) and Mascot phospho-site assignment confidence (bottom) of the two phosphorylated peptides (A and B) detected at S435. Red and yellow signals correspond to the …
Figure 6 with 2 supplements
Effect of full-length NCS-1 on guanine nucleotide exchange factor (GEF) activity of rRic-8A-491.
(A) GTP binding rates were measured by following the increase in rΔN31Gαi1 tryptophan fluorescence following addition of 10 µM GTPγS. Prior to GTPγS addition, rΔN31Gαi1 (1 µM final concentration) …
Figure 6—figure supplement 1
GTPγS binding progress curves.
Representative traces of progress curves in determining nucleotide exchange rates shown in Figure 6. Change in fluorescence intensity is measured at excitation/emission = 295 nm/345 nm, which is …
Figure 6—figure supplement 2
GTP binding rates vs CaCl2 concentration. GTP binding rates of rΔN31Gαi1 vs CaCl2 concentration, after subtraction of the intrinsic rate at 0 µM CaCl2 (black spheres).
GTP binding rates of His-NCS-1/rRic-8A-491 plotted vs CaCl2 concentration after subtraction of intrinsic binding rates of rΔN31Gαi1 at each corresponding CaCl2 concentration (red triangles). In all …
Figure 7
Structural reorganization of Ric-8A for NCS-1 recognition.
(A) The structure of the rRic-8A/G⍺i1 complex (PDB: 6UKT, McClelland et al., 2020). Electrostatic potential surface representation of ARM-HEAT domain (repeats 1–8). The repeat 9 is shown as ribbons. …
Figure 8
Schematic representation of the mechanism of Ric-8A activation regulated by NCS-1.
Step 1: At low Ca2+ concentrations NCS-1 interacts with unphosphorylated Ric-8A (uRic-8A), at the plasma membrane. NCS-1 protects Ric-8A from phosphorylation or Gα subunit binding. Ric-8A ARM-HEAT …
Author response image 1
Comparison of 2D averages and cryo-EM map of the previously presented model and the currently improved one.
(Top left) 2D averages from the previous and current models, generated with the final particles after 3D classification. The old and new cryo-EM maps with docked PDBs from a front (top right), side …
Videos
Video 1
Morph movie explaining the structural rearrangement of Ric-8A HEAT repeat 9 for NCS-1 recognition.
Ric-8A residues 402–429 are shown starting at the Gα-bound and ending at the NCS-1-bound conformations. The view is the same as that in Figure 7A. Side chains are displayed in stick mode. While in …
Tables
Table 1
Diffraction data collection and refinement statistics of hNCS-1/Ric-8A-P crystals.
| Data collection | Structure 1 | Structure 2 | Structure 3 |
|---|---|---|---|
| PDB code | 8ALH | 8AHY | 8ALM |
| Peptide | P2 | P3 | P3 |
| Ions in solution | Mg2+, Ca2+, Na+ | Mg2+, Ca2+, Na+ | Ca2+, Na+ |
| Space group | P41212 | P41212 | P41212 |
| Cell dimensions | |||
| a, b, c (Å) | 56.86, 56.86, 134.61 | 56.64, 56.64, 135.30 | 56.64, 56.64, 134.53 |
| α, β, γ (°) | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 |
| Resolution range (Å) | 52.38–1.86 (1.93-1.86)* [a*, b*=1.846, c*=1.917] | 52.24–1.70 (1.79-1.70)* [a*, b*=1.681, c*=1.891] | 52.20–1.85 (1.94-1.85)* [a*, b*=1.854, c*=1.920] |
| Rpim | 0.044 (0.803) | 0.036 (0.659) | 0.028 (0.616) |
| CC1/2 | 0.998 (0.445) | 0.997 (0.551) | 0.999 (0.467) |
| I/σI | 16.8 (1.2) | 13.5 (1.4) | 15.2 (1.3) |
| Completeness | |||
| Spherical (%) | 92.7 (41) | 88.0 (34.1) | 91.2 (36.1) |
| Ellipsoidal (%) | 94.9 (51.5) | 95.9 (65.4) | 94.0 (45.7) |
| Wilson B-factor | 31.12 | 26.90 | 37.80 |
| Multiplicity | 25.2 (26.5) | 25.5 (27.7) | 8.7 (9.8) |
| Refinement | |||
| Resolution (Å) | 52.38–1.86 | 52.24–1.70 | 52.20–1.85 |
| No. reflections | 18029 | 22008 | 17665 |
| Rwork/Rfree | 19.58/23.13 (26.31/28.49) | 18.64/20.76 (34.65/45.00) | 20.98/25.25 (36.89/46.21) |
| Asymetric unit content | |||
| No. atoms | 3454 | 3403 | 3374 |
| Protein (no. residues) | 171 | 171 | 171 |
| Peptide (no. residues) | 28 | 28 | 28 |
| PEG/GOL | 3/1 | 2/1 | 2/2 |
| Ca2+/Cl-/Mg2+/Na+ ions | 2/1/1/1 | 2/1/1/1 | 2/1/0/2 |
| Water molecules | 141 | 144 | 116 |
| B-factor (Å)2 | 31.27 | 27.28 | 37.18 |
| R.m.s. deviations protein | |||
| Bond lengths (Å) | 0.44 | 0.36 | 0.50 |
| Bond angles (°) | 0.63 | 0.56 | 0.65 |
| R.m.s. deviations peptide | |||
| Bond lengths (Å) | 0.45 | 0.57 | 0.33 |
| Bond angles (°) | 0.70 | 0.63 | 0.61 |
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*
Values in parenthesis are for highest resolution shell.
Table 2
Residues mutated to alanine to validate the NCS-1/Ric-8A PPI interface.
| Residue | Position and interacting residues |
|---|---|
| NCS-1 D37 | Upper part of the crevice. Interacts with Ric-8A R429, which is located at the C-terminal end of R2 helix |
| NCS-1 Y52 | Middle of the crevice. Recognizes Ric-8A L419, which is found at the middle of R2 helix |
| NCS-1 R148 | Bottom of the crevice. Interacts with Ric-8A K408 (N-terminus of R1 helix) and Ric-8A T410 (R1-R2 loop) |
| NCS-1 R151 | Bottom of the crevice. Interacts with Ric-8A K408 (N-terminus of R1 helix) and Ric-8A Y412 (R1-R2 loop, water-mediated H-bond) |
| Ric-8A T410, Y412, N414 | R1-R2 loop. Mediate several water-mediated H-bonds and van der Waals contacts with the bottom surface of NCS-1 crevice |
| NCS-1 W30A | Upper part of the cavity. Important in the recognition of R2 helix. Establish van der Waals interactions with residues such as Ric-8A L424 and M425 |
| Ric-8A L424 and M425 | C-terminal part of helix R2. Interact with NCS-1 W30 and the hydrophobic environment that surrounds these residues |
Table 3
The Ca2+-dependent affinity of full-length NCS-1 for Ric-8A-P3 peptide.
Calculated apparent Kd and standard error of the mean (SEM) using biolayer interferometry. Three independent experiments were performed at each [Ca2+].
| [Ca2+] (nM) | ||||
|---|---|---|---|---|
| 0 | 250 | 375 | 425 | |
| Kd (μM) | 140 | 344 | 381 | 98620 |
| SEM | 29 | 14 | 18 | 16310 |
Table 4
Thermodynamic parameters of Ca2+ binding to full-length hNCS-1 in the presence of K+ or Na+.
| C (150 mM) | Kd1 (nM) | ΔH1 (kcal/mol) | Kd2 (nM) | ΔH2 (kcal/mol) | Kd3 (nM) | ΔH3 (kcal/mol) |
|---|---|---|---|---|---|---|
| Na+ | 265±6 | –7.7±0.1 | 758±16 | 3.0±0.1 | 379±17 | –9.1±0.3 |
| K+ | 165.6±0.3 | –7.66±0.01 | 362.3±0.6 | 1.00±0.01 | 253±1 | –9.44±0.01 |
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Subscripts 1, 2, and 3 correspond to sites 1, 2, and 3, respectively.
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21* | Invitrogen | Cat # C601003 | |
| Strain, strain background (E. coli) | BL21(DE3) pLysS | Novagen | Cat # 70236 | |
| Strain, strain background (E. coli) | Rosetta2 pLysS | Novagen | Cat # 71403 | |
| Strain, strain background (E. coli) | WK6 cells | McClelland et al., 2020 | Cat # 47078 | |
| Strain, strain background (E. coli) | BL21 CodonPlus (DE3) RIPL | McClelland et al., 2020 | Cat # 230280 | |
| Cell line (human) | HEK293 | ATCC | Cat # CRL-1573 | |
| Antibody | Mouse monoclonal anti-V5 | Invitrogen | Cat # R960-25 | 1:5000 |
| Antibody | Rabbit polyclonal anti-NCS-1 | Cell Signaling | Cat # 8237S | 1:2000 in WB 1:500 in IP |
| Antibody | Anti-mouse antibody TrueBlot secondary | Rockland | Cat # 18-8817-30 | 1:5000 |
| Recombinant DNA reagent | Human NCS-1 (full-length) in pETDuet vector | Canal-Martín et al., 2019 | N/A | |
| Recombinant DNA reagent | Human NCS-1ΔH10 in pETDuet vector | This work | N/A | Stop codon after residue P177 for NCS-1ΔH10 construct |
| Recombinant DNA reagent | Human His(6)-NCS-1 in pET28a+vector | This work | N/A | His-tagged NCS-1 version in pET28a+ vector |
| Recombinant DNA reagent | Human His(6)-NCS-1 (full-length) in pETDuet vector | This work | N/A | His-tagged NCS-1 version in pETDuet vector |
| Recombinant DNA reagent | Rat His(6)-Ric-8A(1-452) in pET28a vector | Thomas et al., 2011 | N/A | |
| Recombinant DNA reagent | Rat His(6)-Ric-8A(1-423) in pET28a vector | This work | N/A | Ric-8A(1-423) truncated version of Rat His(6)-Ric-8A(1-452) in pET28a vector |
| Recombinant DNA reagent | Rat His(6)-Ric-8A(1-432) in pET28a vector | This work | N/A | Ric-8A(1-432) truncated version of Rat His(6)-Ric-8A(1-452) in pET28a vector |
| Recombinant DNA reagent | Rat His(6)-Ric-8A(1-491) in pET28a vector | Thomas et al., 2011 | N/A | |
| Recombinant DNA reagent | Rat GST-ΔN31Gα in a pDest15 vector | McClelland et al., 2020 | N/A | |
| Recombinant DNA reagent | Human Ric-8A deletion construct ending at G424 (hRic-8A-424) in nV5-pCDNA3.1 plasmid | This work | N/A | hRic-8A-G424 construct version of human Ric-8A in nV5-pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human Ric-8A deletion construct ending at G433 (hRic-8A-433) in nV5-pCDNA3.1 plasmid | This work | N/A | hRic-8A-G433 construct version of human Ric-8A in nV5-pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human Ric-8A full-length mutant (S436A, T441A) in nV5-pCDNA3.1 plasmid | This work | N/A | Phosphorylation mutant version |
| Recombinant DNA reagent | Human Ric-8A in nV5-pCDNA3.1 plasmid | Mansilla et al., 2017 | N/A | |
| Recombinant DNA reagent | Human Ric-8A mutant (T411A, Y413A, N415A) in nV5-pCDNA3.1 plasmid | This work | N/A | T411A, Y413A, N415A mutant version of human Ric-8A in nV5-pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human Ric-8A mutant (L425A, M426A) in nV5-pCDNA3.1 plasmid | This work | N/A | L425A, M426A, mutant version of human Ric-8A in nV5-pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human NCS-1 in pCDNA3.1 plasmid in pCDNA3.1 plasmid | Mansilla et al., 2017 | N/A | |
| Recombinant DNA reagent | Human NCS-1 mutant (D37A, Y52A) in pCDNA3.1 plasmid | This work | N/A | D37A, Y52A mutant version of human NCS-1 in pCDNA3.1 plasmid in pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human NCS-1 mutant (R148A, R151A) in pCDNA3.1 plasmid | This work | N/A | R148A, R151A mutant version of human NCS-1 in pCDNA3.1 plasmid in pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human NCS-1 mutant (D37A, R148A, R151A) in pCDNA3.1 plasmid | This work | N/A | D37A, R148A mutant version of human NCS-1 in pCDNA3.1 plasmid in pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human NCS-1 mutant (D37A, Y52A, R148A, R151A) in pCDNA3.1 plasmid | This work | N/A | D37A, Y52A, R148A, R151A mutant version of human NCS-1 in pCDNA3.1 plasmid in pCDNA3.1 plasmid |
| Recombinant DNA reagent | Human NCS-1 mutant (W30A) in pCDNA3.1 plasmid | This work | N/A | W30 mutant version of human NCS-1 in pCDNA3.1 plasmid in pCDNA3.1 plasmid |
| Peptide, recombinant protein | Casein kinase II | New England Biolabs | Cat # P6010L | |
| Peptide, recombinant protein | Ric-8A P1 peptide (400-423) | GenicBio | N/A | |
| Peptide, recombinant protein | Ric-8A P2 peptide (400-429) | GenicBio | N/A | |
| Peptide, recombinant protein | Ric-8A P3 peptide (400-432) | GenicBio | N/A | |
| Chemical compound, drug | ATP | New England Biolabs | Cat # P0756S | |
| Chemical compound, drug | Water for UHPLC-MS LiChrosolv | Merck | Cat # 1037282002 | |
| Chemical compound, drug | Guanosine 5’-[g-thio]triphosphate | Sigma | Cat # G8634-10MG | |
| Software, algorithm | ImageJ | Schneider et al., 2012 | https://imagej.net/software/imagej/ | |
| Software, algorithm | GraphPad Prism | GraphPad Software, Inc, USA; GraphPad Prism, 2023 | https://www.graphpad.com/features | |
| Software, algorithm | AutoPROC | Vonrhein et al., 2011 | https://www.globalphasing.com/autoproc/manual/autoPROC1.html | v1.1.7 |
| Software, algorithm | Phaser | McCoy et al., 2007 | https://www.ccp4.ac.uk/html/phaser.html | v2.7.0 |
| Software, algorithm | Phenix | Adams et al., 2010 | https://www.phenix-online.org/ | v1.19.2_4158 |
| Software, algorithm | COOT | Emsley and Cowtan, 2004 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | 0.9.8 |
| Software, algorithm | Molprobity | Williams et al., 2018 | http://molprobity.biochem.duke.edu/ | v4.5.2 |
| Software, algorithm | CCP4 | Winn et al., 2011 | https://www.ccp4.ac.uk/ | v8.0 |
| Software, algorithm | PISA Server | Krissinel and Henrick, 2007 | https://www.ebi.ac.uk/pdbe/pisa/ | 1.48 |
| Software, algorithm | PyMol | Schrödinger, 2015 | https://pymol.org/2/ | v1.8.6.0 |
| Software, algorithm | Tycho NT.6 software | NanoTemper | https://nanotempertech.com | Tycho |
| Software, algorithm | Mascot Server | Matrix Science | https://www.matrixscience.com/ | |
| Software, algorithm | KaleidaGraph Data Analysis Program | Synergy Software | https://www.synergy.com/ | |
| Software, algorithm | AFFINImeter | AFFINImeter | https://www.affinimeter.com | |
| Other | Atomic Coordinates and Structure Factors NCS-1/Ric-8A-P2 complex | PDB | Structure 1 | 8ALH |
| Other | Atomic Coordinates and Structure Factors NCS-1/Ric-8A-P3 complex | PDB | Structure 2 | 8AHY |
| Other | Atomic Coordinates and Structure Factors NCS-1/Ric-8A-P3 complex | PDB | Structure 3 | 8ALM |
| Other | Hi Trap Phenyl FF hydrophobic column | Cytiva | 17519301 | Purification column |
| Other | Anion exchange HP Q column | Cytiva | 17115301 | Purification column |
| Other | Nickel-affinity column, HisTrap FF | Cytiva | 17525501 | Purification column |
| Other | Ni2+-chelated Sepharose HP beads | Cytiva | 17526801 | Purification column |
| Other | HiLoad 16/600 Superdex 200 pg | Cytiva | 28989335 | Purification column |
| Other | Source 15Q column | Cytiva | 17094701 | Purification column |
| Other | Superdex 200 HR 10/300 column | Cytiva | GE17-5175-01 | Purification column |
| Other | Tycho NT.6 instrument | NanoTemper | https://nanotempertech.com/tycho/ | Nano-DSF equipment |
| Other | Tycho capillaries | NanoTemper | Cat # TY-C001 | Nano-DSF capillaries |
| Other | VP-ITC microcalorimeter | GE Healthcare | https://www.malvernpanalytical.com/en/products/product-range/microcal-range | ITC equipment |
| Other | BLItz system | ForteBio | BLItz from ForteBio | BLI equipment |
| Other | Ni-NTA biosensors | Sartorius | CA89413-836 | BLI biosensors |
| Other | Protein-G-Sepharose | Sigma-Aldrich | CAT # P3296-1ML | Antibodies purification |
