Dynamic Response of the C2 Domain of Protein Kinase Cα to Ca2+ Binding Krystal A. Morales, Yuan Yang, Taylor R. Cole, Tatyana I. Igumenova  Biophysical Journal  Volume 111, Issue 8, Pages 1655-1667 (October 2016) DOI: 10.1016/j.bpj.2016.09.008 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Host protein context and structural features of C2α. (A) Linear diagram of cPKCs, showing their domain structure. (B) Crystal structure of C2α complexed to Ca2+ and PtdIns(4,5)P2 (PDB: 3GPE). The LRC region is the β3-β4 hairpin, shown in red. The LRC is the interaction site of the C2 domain with PtdIns(4,5)P2. Residues implicated in intramolecular interactions with the C1A domain are in yellow. (C) Top view of the Ca2+ coordination site of C2α. The Ca2+ ions have all-oxygen coordination spheres, with ligands provided by the carboxyl oxygens of aspartic acids and the carbonyl oxygens of M186 (Ca2), W247 (Ca1), and R252 (Ca3). The loops are represented as ribbons. To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Site 3 of C2α is populated in solution at high Ca2+ concentrations. (A) Coordination geometry of Ca3 in the structure of C2α complexed to PtdIns(4,5)P2 (PDB: 3GPE). (B) Expansions of the 15N-1H HSQC spectra showing the response of the N-H groups of two amino acids, T250 and D248, to binding Ca3. (C) Representative NMR-detected binding curves. Kd,app was obtained from the global fit (solid lines) of nine CMBL3 residues. To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Subnanosecond dynamics of the C2α backbone in different states of metal ligation. (A) Generalized order parameter S2NH plotted against the primary structure for apo C2α, C2α⋅Can, and C2α⋅Pb. The S2NH for the four Trp side chains in C2α are shown in the adjacent plot. The green box highlights the LRC region. (B) S2NH mapped onto the structures of apo C2α (PDB: 3RDJ). Residues for which data were not available are colored gray. (C) Residue-specific heteronuclear {1H}-15N nOe and spectral density values, the latter determined at 0.87 × ωH = 521.8 MHz, plotted against the primary structure of the loop-containing regions. To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Evidence for microsecond-timescale dynamics in the apo C2α. (A) Transverse relaxation rate constants, R2,CPMG, determined at 14.1 T, plotted against the primary structure for the apo C2α, C2α⋅Can, and C2α⋅Pb states. The total Ca2+ and Pb2+ concentrations were 10 and 0.41 mM in the C2α⋅Can and C2α⋅Pb samples, respectively. (B and C) Differences between the R2,CPMG values, ΔR2,CPMG, were calculated for pairs of states apo C2α and C2α⋅Pb (B) and apo C2α and C2α⋅Can (C). The inset in (B) shows the coordination geometry of Pb2+ bound to the high-affinity site (PDB: 3TWY). To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 The chemical-exchange process is Ca2+-dependent and involves residues in the N- and C-terminal regions. (A) 15N rc-CPMG dispersion curves in the apo and Ca2+-bound C2α are shown for the four residues with the largest dispersion amplitudes. The apo and Ca2+ data were collected at 14.1 T and 11.7 T, respectively. For Ca2+ data, solid lines are the fits to the Carver-Richards equation (62). (B) Residues with quantifiable exchange behavior mapped onto the structure of C2α (PDB: 3GPE). |ΔωN| are the absolute values of the 15N chemical-shift differences between the exchanging conformers obtained from fitting the dispersion data. The residue identities, along with the fit parameters, are given in Tables 1 and S3. (C) Ca2+ dependence of the 15N R2,HE values obtained at [Ca2+] ranging from 1.75 to 87.5 mM. The data were collected at 14.1 T. (D) Plots of selected C2α residues showing an increase in R2,HE values as a function of Ca2+ concentration. To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 The R159G mutation alters the conformation and dynamics of C2α through perturbation of the H-bonding network that brings C- and N-termini in spatial proximity. (A) The 1HN-15N CSP plot of R159G versus wild-type C2α. Perturbations due to mutation are located throughout the protein, including in strand β7, which immediately follows CMBL3, and the four-residue segment that precedes helix H3; the β-bridge Lys278 is labeled with “B.” (B) ΔR2,CPMG values calculated using the data at 14.1 T. A large increase in the microsecond dynamics is observed in the C-terminal region of the R159G variant. (C) Comparison of the 15N rc-CPMG relaxation dispersion curves obtained for Ca2+-complexed R159G and C2α at 14.1 T and [Ca2+] = 10 mM. The R159G mutation eliminates the chemical-exchange process present in Ca2+-complexed wild-type C2α. (D) The hydrogen bond between the Hη2 of the Arg159 side chain and the carbonyl oxygen of Met256 connects the N-terminus to the β7 strand of the protein core. (E) Hydrogen-bonding network in C2α that stabilizes C- and N-terminal regions. The key element is the three-residue segment comprised of Lys276, Leu277, and Leu278. (D) and (E) were generated using the crystal structure of Pb2+-complexed C2α (PDB: 3TWY) (14). To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Self-association of C2α. (A) Intermolecular 1H Γ2 values (open circles) plotted against the primary structure of C2α. The NMR sample was a 1:1 mixture of diamagnetic [U-15N] C2α and paramagnetic C2α at natural abundance (“NA”). Ca2+ concentration in the sample was 10.6 mM. The red solid line is the 1H Γ2 values back-calculated using the structure of the C2β dimer (PDB: 1A25). (B) Systematic Ca2+-dependent increase of R2,CPMG values in C2α. The R2,CPMG data sets (diamonds) were trimmed to remove residues that showed Ca2+-dependent chemical exchange. Open circles represent the median R2,CPMG values. (C) Orientation and hydrogen-bonding partners of the Arg159 side chain in the C2α monomer (PDB: 3TWY) and C2β dimer (PDB: 1A25). The monomer and dimer are color-coded tan and gray, respectively. To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 8 Models of chemical exchange. (A) The kinetic scheme of monomeric exchange in solution. (B) Schematic illustration of how destabilization of the terminal regions (labeled “N” and “C”) of Ca2+- and membrane-bound C2 in cPKCs may facilitate the release of other membrane binding modules, C1A and C1B. Their interaction with membrane-embedded diacylglycerol is required for PKC activation. Ca2+ ions are shown with cyan spheres; H3 is α-helix 3. To see this figure in color, go online. Biophysical Journal 2016 111, 1655-1667DOI: (10.1016/j.bpj.2016.09.008) Copyright © 2016 Biophysical Society Terms and Conditions