Backbone and side chain dynamics of mutant calmodulin-peptide complexes

Abstract

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) has been explored by characterizing structural and dynamics effects of mutants of calmodulin in complex with a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp). Four CaM mutants were examined: D95N and D58N, located in Ca2+-binding loops; and M124L and E84K, located in the target domain-binding site of CaM. Three of these mutants have altered allosteric coupling either between Ca2+-binding sites (D58N and D95N) or between the target- and Ca2+-binding sites (E84K). The structure and dynamics of the mutant calmodulins in complex with smMLCKp were characterized using solution NMR. Analysis of chemical shift perturbations was employed to detect largely structural perturbations. 15N and 2H relaxation was employed to detect perturbations of the dynamics of the backbone and methyl-bearing side chains of calmodulin. The least median squares method was found to be robust in the detection of perturbed sites. The main chain dynamics of calmodulin are found to be largely unresponsive to the mutations. Three mutants show significantly perturbed dynamics of methyl-bearing side chains. Despite the pseudosymmetric location of Ca2+-binding loop mutations D58N and D95N, the dynamic response of CaM is asymmetric, producing long-range perturbation in D58N and almost none in D95N. The mutations located at the target domain-binding site have quite different effects. For M124L, a local perturbation of the methyl dynamics is observed, while the E84K mutation produces a long-range propagation of dynamic perturbations along the target domain-binding site.

Figure 1

Chemical shift-based identification of structural perturbations in the CaM-smMLCKp complex introduced by the D95N mutation in calmodulin. (A) Standardized residuals calculated using one-dimensional LMS (red) and LS (yellow) analyses of ¹⁵N-¹H cross-peak displacements Δ in the D95N-smMLCKp complex. Dotted lines represent the ± 2.5 interval of the standardized residuals. The amide groups that have absolute values of the residuals greater than 2.5 are considered to be chemical shift outliers. (B) ¹⁵N-¹H cross-peak displacement Δ in the D95N-smMLCKp complex. The amide groups that are identified as outliers by the LMS analysis are colored blue. Positions of the calcium-binding loops are indicated by the solid bars. 15 and 4 outliers were identified by LMS and conventional LS analyses, respectively.

Figure 2

Chemical shift-based identification of structural perturbations in the CaM-smMLCKp complex introduced by the E84K mutation in calmodulin. (A) Standardized residuals calculated using one-dimensional LMS (red) and LS (yellow) analyses of ¹⁵N-¹H cross-peak displacements Δ in the E84K-smMLCKp complex. Dotted lines represent the ± 2.5 interval of the standardized residuals. The amide groups that have absolute values of the residuals greater than 2.5 are considered to be chemical shift outliers. (B) ¹⁵N-¹H cross-peak displacement Δ in the E84K-smMLCKp complex. The amide groups that are identified as outliers by the LMS analysis are colored blue. Positions of the CaM helices are indicated by the solid bars. 22 outliers are identified by LMS, compared to only 7 outliers identified by conventional LS. (C) Squared displacement of backbone atoms between the wild-type CaM-smMLCKp (PDB code 1cdl) and E84K-RS20 (PDB code 1vrk) structures mapped onto the structure of CaM-smMLCKp. The structure of the peptide is not shown. A blue-white-red color gradient is used to represent the range of squared atom displacements from 0 to 3 Ų. The average R.M.S.D. for backbone atoms of residues 5-146 of calmodulin is 0.94 Å. The results of superposition of the CaM-smMLCKp and E84K-RS20 structures are in general consistent with amide chemical shift data, where helix E and the loop between helices E and D can be identified as the most perturbed protein regions.

Figure 3

Chemical shift-based identification of structural perturbations in the CaM-smMLCKp complex introduced by various mutations in calmodulin. Shown are superpositions of the ¹³C-¹H chemical shift correlation maps for the wild-type CaM-smMLCKp (black) and E84K-smMLCKp (red); wild-type CaM-smMLCKp (black) and D58N-smMLCKp (green). Many methyl groups throughout the E84K-smMLCKp complex, including all methionines, experience large chemical shift changes as a result of the mutation. D58N-smMLCKp is a more moderate case, where chemical shift perturbations are localized to Ca²⁺-binding loops I and II and helix C.

Figure 4

Identification of dynamical perturbations in the CaM-smMLCKp complex introduced by various mutations in calmodulin. Difference between methyl group symmetry axis order parameters $(\mathrm{\Delta }{O}_{\mathrm{axis}}^2)$ of the mutant and wild-type smMLCKp complexes. The errors bars are calculated as a sum of individual absolute errors of the corresponding mutant and wild-type order parameters. Outliers identified by LMS analysis are labeled with asterisks. Three patterns of perturbation in the side chain methyl dynamics are observed: almost no perturbation in D95N-, mostly local in M124L-, and long-range in D58N- and E84K-smMLCKp complexes. Note that the vertical and horizontal scales in the M124L graph are different from those in the other three graphs.

Figure 5

Methyl axis order parameters, $O_{\mathrm{axis}}^2$, of the E84K-smMLCKp (A) and D95N-smMLCKp (B) complexes versus those of the wild-type CaM-smMLCKp complex. The least median squares (LMS) fit is shown with a solid line. Outliers identified by the LMS analysis are colored red. The value of the intercept was not forced to zero in the LMS analysis to account for possible systematic differences caused by the uncertainties in global correlation time. Since the average value of the $O_{\mathrm{axis}}^2$ is the same for all mutant and wild-type complexes within the experimental error, forcing the constant to zero produced an essentially identical set of outliers.

Figure 6

Standardized residuals ${\widehat{\widehat{r}}}_i$ mapped onto the three-dimensional structure of CaM-smMLCKp (PDB code 1cdl) using a blue-white-red color gradient to represent the range of ${\widehat{\widehat{r}}}_i$ from −5.0σ to +5.0σ. The values of scale estimate σ are given at the bottom of each panel. Mutated residues are shown in stick representation and are labeled. The peptide structure and calcium atoms are displayed in green. In C, the $\widehat{\widehat{r}}$ value of L124δ is mapped onto the Cɛ carbon of M124 using an average for the two methyl groups of L124. The individual $\widehat{\widehat{r}}$ values for the two L124 methyl groups are given in Table 1. In D58N-smMLCKp, two methyl groups with perturbed τ_e are shown in yellow.

Figure 7

A cartoon of the E84K-RS20 structure (PDB code 1vrk) showing the relative position of calmodulin helix E and the RS20 peptide. The structures of helix E in the wild-type and E84K complexes are superimposed and shown in purple and orange, respectively. The mutated side chain is shown in stick representation and is labeled. In the E84K-RS20 complex, the side chain of K84 rotates away from the peptide. The values of standardized residuals $\widehat{\widehat{r}}$ are mapped onto methyl carbons using the same color gradient as in Figure 6. Since in VU-1 CaM position 71 is occupied by leucine, the standardized residual $\widehat{\widehat{r}}$ of M71ɛ is mapped onto the leucine δ methyl groups. The peptide side chains that are within 5 Å of the perturbed methyls are shown in ball-and-stick representation.