SUPPLEMENTARY MATERIAL Materials and Methods Circular dichroism (CD) spectroscopy. Far ultraviolet (UV) CD spectra of apo- and holo- CaM and the CaM mutants were recorded on a Jasco J-715 spectropolarimeter at 20 o C in 20 mm HEPES ph 8.0, 100 mm KCl, 1 mm MgCl 2 and 200 M EGTA or 1 mm CaCl 2, as appropriate. Spectra are presented as the CD absorption coefficient calculated on a mean residue weight (mrw) basis. Ca 2+ binding measurements. Stoichiometric Ca 2+ dissociation constants for wild type and mutant CaMs were determined from Ca 2+ titrations of the apo proteins (23-27 M) performed in the presence of the chromophoric chelator 5,5 -Br 2 BAPTA (25 M) (26,27). Ca 2+ -binding to the chelator dye was measured in the presence or absence of CaM and the bound fraction was measured by a decrease in absorption compared to the free dye as CaM and its mutants competed for Ca 2+ with the chelator (Figure S2). Measurements were made at 20 o C in 10 mm Tris.HCl ph 8 and 100 mm KCl. Under these conditions, the Ca 2+ binding constant of 5,5 - Br 2 BAPTA was 5.7 x 10 5 M -1 (27). Three separate titrations were performed on each protein sample, and the values for the individual binding constants were obtained from nonlinear least-squares fits directly to the experimentally observed titration curves. The binding data were fitted to a model in which the higher affinities were assigned to the two C lobe sites (K d 3 and K d 4) and the lower affinities to the two N lobe sites (K d 1 and K d 2) of CaM (26,27). From the measured K d 1, K d 2, K d 3 and K d 4, values, an overall K d1 was derived for convenience, for wild type and mutant CaMs (as described in Materials and Methods, Theory section and Table S2), to be used in Scheme 1 and Reaction 1 below. Ca 2+ dissociation kinetics. Stopped-flow kinetic measurements of Ca 2+ dissociation were carried out using quin 2 (Molecular Probes) and a Hi-Tech Scientific SF-61DX2 stopped-flow system as previously described (30) and briefly: fluorescence excitation was set to 320 nm with 1 nm slit width and fluorescence emission from quin 2 was collected using a 530 nm cutoff filter. The assay solution contained 50 mm K + -PIPES ph 7.0, 100 mm KCl, 2 mm MgCl 2. 90 µm quin 2 in assay solution with no added Ca 2+ was mixed with 3 µm wild type or mutant CaM in a 50 µm Ca 2+ -containing buffer solution. In parallel sets of experiments 5 M CaMKII 294-309 peptide from the Ca 2+.CaM binding domain of CaMKII was included (mixing chamber concentrations). Care was taken that all protein components were free of any Ca 2+ chelator. 1
SUPPLEMENTARY TABLES Table S1 Far UV CD properties of CaM mutants. Molar CD absorption coefficients, (M -1 cm -1 ) at 222 nm were determined for mutant CaMs and compared with those for wild type, in the absence and presence of Ca 2+, as a measure of secondary structure content. Table S2 Macroscopic Ca 2+ dissociation constants of wild type and mutant CaMs. Each value represents the mean of three sets of measurements, error is S.D. K d1 represents the overall Ca 2+ dissociation constant for CaM corresponding to K d1 = {1/(K1*K2*K3*K4)} 1/4 (where K1 denotes the association constant for Ca 2+ binding site 1, and so on) for wild type CaM and to the appropriate sites and number of sites for each mutant e.g. for K d1 for CaM1= {1/(K2*K3*K4)} 1/3. Table S3 Ca 2+ dissociation rate constants of CaM mutants and their CaMKII peptide complexes. Stopped-flow kinetic measurements were carried out as described in Materials and Methods. n indicates the number of records averaged, S.E.M. values are the standard error of the mean of n experiments. k off1 and k off2 (s -1 ) represent the rate constants and A1 and A2 correspond to the amplitudes of the first and second phases (when observed). Amplitudes are expressed as % signal change observed in the experiments. SUPPLEMENTARY FIGURES Fig. S1 Far-UV CD spectra of CaM mutants in the presence and absence of Ca 2+. Spectra of the apo (1, red line) and holo (2, blue line) forms of CaM and the CaM mutants A) CaM1, B) CaM2, C) CaM3, D) CaM4, E) CaM12, F) CaM34 and G) wild type CaM. Fig. S2 Ca 2+ titrations of CaM mutants. A) CaM1, B) CaM2, C) CaM3, D) CaM4, E) CaM12, F) CaM34 and G) wild type CaM. Panel G shows titration of 25 M 5,5 -Br 2 BAPTA in the absence of protein (blue squares) and in the presence of 25 M wild type CaM (red label). The fitted curves for these displayed as grey lines are used to overlay the titration data for each mutant to indicate the position of the dye and wild type CaM for comparison. The concentration of the mutants CaMs was in the range of 23-27 M. Fig. S3 Kinetic parameters of CaMKII activity stimulated by wild type and mutant CaMs, with respect to calmodulin. Steady-state activity measurements were carried out as described in Materials and Methods, syntide 2 concentration was 50 M, free Ca 2+ concentration was set to 60 M. The free Ca 2+ concentration was calculated using the overall K d1 values for CaM and its mutants (Suppl. Table S2) as described in Materials and Methods, except for CaM34 for which free [Ca 2+ ] of 100 M was maintained, CaMKII was 50 nm, ATP was 1 mm. The panels show A) CaM1, ( ) and CaM2, ( ); B) CaM3, ( ) and CaM4, ( ); C) CaM12, ( ), CaM34, ( ) and wild type CaM, ( ). The error bars represent the standard error of the mean (S.E.M.) from three sets of data. 2
Table S1 Far UV CD properties of CaM mutants. Protein (- Ca 2+ ) (+ Ca 2+ ) M -1 cm -1 M -1 cm -1 (+ Ca 2+ )/ (- Ca 2+ ) CaM -4.29 0.09-5.32 0.11 1.24 0.04 CaM1-4.22 0.08-5.03 0.10 1.19 0.03 CaM2-4.19 0.08-4.91 0.10 1.17 0.03 CaM3-4.27 0.09-4.91 0.10 1.15 0.03 CaM4-4.33 0.08-4.94 0.10 1.14 0.03 CaM12-4.37 0.09-4.85 0.10 1.11 0.03 CaM34-4.16 0.08-4.41 0.09 1.06 0.03 CaM1234-4.19 0.08-4.23 0.08 1.01 0.03 3
Table S2 Ca 2+ binding properties of wild type and mutant CaMs K d 1 K d 2 K d 3 K d 4 K d1 CaM 52.5 0.8 5.6 0.4 6.9 0.5 0.4 0.6 5.5 CaM1 239.9 0.5 4.8 0.9 0.5 0.8 8.1 CaM2 97.7 0.6 6.9 0.4 0.4 0.5 6.4 CaM3 30.2 0.6 17.4 0.6 16.2 0.5 20.4 CaM4 12.0 0.5 9.5 0.6 21.4 0.9 13.4 CaM12 6.3 0.8 0.9 0.5 2.3 CaM34 70.8 0.8 3.7 0.5 16.0 It must be noted that the affix for the macroscopic dissociation constants does not assign a specific Ca 2+ binding site of CaM. K d 1 and K d 2 refer to the two N lobe sites, though not specifically to either site 1 or site 2, and K d 3 and K d 4 describe binding to the two C lobe sites on the same terms. Overall K d1 was calculated as follows: K d1 = (K d 1 K d 2 K d 4) 1/n, where n is the number of Ca 2+ sites present in the mutant. 4
Table S3 Ca 2+ dissociation kinetics of CaM mutants and their complex with CaMKII 294-309 k off1 s -1 S.E.M A1 S.E.M k off2 s -1 S.E.M A2 S.E.M n WT CaM 11.4 0.7 6.4 0.2 9 WT CaM + CaMKII 294-309 0.3 0.1 12.3 0.4 6 CaM1 12.5 1.9 5.4 0.5 9 CaM1 + CaMKII 294-309 1.0 0.0 6.7 0.2 5 CaM2 11.2 1.4 5.7 0.5 9 CaM2 + CaMKII 294-309 0.53 0.02 7.1 0.1 5 CaM3 242 76 5.0 1.1 9 CaM3 + CaMKII 294-309 5.1 0.2 10.0 0.2 5 CaM4 289 99 3.5 0..5 9 CaM4 + CaMKII 294-309 3.3 0.1 5.7 0.2 5 CaM12 11.2 1.9 6.2 0.5 8 CaM12 + CaMKII 294-309 5.3 0.6 6.3 0.4 0.6 0.1 2.6 0.28 8 CaM34 214 210 3.4 2.7 8 CaM34 + CaMKII 294-309 5.1 0.3 6.8 0.3 8 CaM1234 N.D. N.D. 9 CaM1234 + CaMKII 294-309 N.D. N.D. 9 5
Figure S1 6
Figure S2 7
Figure S3 8