Figure 1.
Amino acid sequence alignment of GCAP1 with various NCS proteins.
Secondary structural elements are indicated schematically. The four EF-hands (EF1, EF2, EF3 and EF4) are underlined. Residues mutated in EF4mut (D144N/D148G) are indicated in red. Residues at the domain interface (V77 – L97) that have broadened NMR resonances are shown in italics.
Figure 2.
NMR spectra of activator vs. inhibitor forms of GCAP1.
Two-dimensional (1H-15N HSQC) NMR spectra of 15N-labeled wildtype Ca2+-saturated GCAP1 (A) and EF4mut (B). Spectra were obtained at 37 °C in the presence of 40 mM octylglucoside. Downfield resonances (~10.5 ppm) are assigned to conserved glycine residues in each Ca2+-bound EF-hand loop. For Ca2+-saturated GCAP1 (A), downfield peaks assigned to G69, G105 and G149 indicate Ca2+ is bound at EF2, EF3 and EF4. For EF4mut (B), downfield peaks assigned to G69 and G105 indicate Ca2+ is bound at EF2 and EF3 in EF4mut. Sequence specific resonance assignments for Ca2+-saturated GCAP1 were determined previously [31].
Figure 3.
GCAP1 forms a dimer in solution.
15N NMR relaxation data are shown for EF4mut. Spin-lattice relaxation rate constants (A) and spin-spin relaxation rate constants (B) are plotted as a function of residue number. All data were measured at 60.81 MHz 15N frequency and 37 °C. (C) Size-exclusion chromatography (SEC) elution profiles are shown for GCAP1 wildtype (solid line) and V77E (dotted line). The protein concentration was 200 μM for the samples analyzed by SEC.
Figure 4.
NMR chemical shift mapping for GCAP1.
(A) Amide chemical shift differences between EF4mut and Ca2+-saturated wildtype (CSD = {(HNA – HNI)2 + (15NA – 15NI)2}1/2 , where “A” and “I” designate activator and inhibitor states) plotted as a function of residue number. (B) CSD values from part “A” are mapped onto the GCAP1 crystal structure (2R2I). Residues with the largest chemical shift difference (CSD > 0.12 ppm) are highlighted in red, intermediate chemical shift differences (0.06 < CSD < 0.12) shown in yellow, and smallest chemical shift differences (CSD < 0.06) shown in green. Unassigned residues are colored gray and the myristoyl group is orange.
Figure 5.
Structural characterization of GCAP1 mutants at the domain interface.
(A) Overlay of 15N-1H HSQC spectra of V77E (red), L82E (blue), WT (black) and W94F (green) indicate that each mutant is properly folded and structurally intact. The mutant spectra look similar to that of wildtype particularly for residues in structured regions. Minor spectral differences are observed for exposed residues in unstructured regions, most likely due to small differences in solvent conditions. (B) Expanded view of 15N-1H HSQC downfield region for GCAP1 mutants: V77E (red), L82E (blue), WT (black) and W94F (green). Three downfield NMR peaks assigned to G69, G105 and G149 indicate Ca2+ is bound functionally at EF2, EF3 and EF4 for each mutant.
Figure 6.
Schematic model of conformational changes in GCAP1 caused by Ca2+-binding at EF4.
(A) Structural model of EF4mut activator state was generated by homology modeling using the NMR structure of recoverin (1jsa) that contains Ca2+ bound at EF2 and EF3 [23]. The four EF-hands are colored green (EF1), red (EF2), cyan (EF3) and yellow (EF4), and bound Ca2+ is orange. (B) The crystal structure of Ca2+-saturated GCAP1 (2r2i) shows key hydrophobic residues at the domain interface are solvent exposed in the Ca2+-bound inhibitor state. The Ca2+-dependent rearrangement of the W94 side-chain at the domain interface might control the switching between activator and inhibitor states. The N-terminal myristoyl group (magenta) is sequestered inside the protein in both structures.