Fast methionine-based solution structure determination of calcium-calmodulin complexes

Abstract

Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca(2+)-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaM's backbone conformation and a structural plasticity in CaM's domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant. Although RDCs can define CaM domain orientation in the complex, they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs. Here we employ a specific isotope labeling strategy in which the role of methionine in CaM-peptide interactions is exploited to collect these critical NOEs. By (1)H, (13)C-labeling the methyl groups of deuterated methionine against a (2)H, (12)C background, we can acquire a (13)C-edited NOESY characterized by simplified, easily analyzable spectra. Together with measured CaM backbone H(N)-N RDCs and intrapeptide NOE-based distances, these intermolecular NOEs provide restraints for a low temperature torsion-angle dynamics and simulated annealing protocol used to calculate the complex structure. We have applied our method to a CaM complex previously solved through X-ray crystallography: Ca(2+)-CaM bound to the CaM kinase I peptide (PDB code: 1MXE). The resulting structure has a backbone RMSD of 1.6 Å to that previously published. We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome. In addition to having application for fast complex structure determination, this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully (13)C, (15)N-labeled CaM fails.

Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

“…Sample Preparation-Unlabeled and isotopically enriched CaM was recombinantly expressed in Escherichia coli BL21(DE3) cells containing the pET30b(ϩ) expression vector as described previously (15). For isotope labeling, minimal medium containing 15 1 H-␣,⑀-13 C-⑀-2 H-labeled methionine was added to the culture 1 h prior to induction with isopropyl ␤-D-1-thiogalactopyranoside (15).…”

“…For isotope labeling, minimal medium containing 15 1 H-␣,⑀-13 C-⑀-2 H-labeled methionine was added to the culture 1 h prior to induction with isopropyl ␤-D-1-thiogalactopyranoside (15). CaM samples were purified to homogeneity by Ca 2ϩ -dependent phenyl-Sepharose chromatography.…”

“…Resonance assignments of the backbone and side chain atoms for CaM in complex with LSELl (L-selectin long peptide) were obtained using through-bond heteronuclear scalar couplings with the standard pulse sequences (15). For assignment of the side chain methyl group of the methionines, three-dimensional HMBC and LRCH experiments that record the long range correlations between the H⑀/C⑀ and H␥/C␥ atoms were used (16) Table S1).…”

“…For assignment of the side chain methyl group of the methionines, three-dimensional HMBC and LRCH experiments that record the long range correlations between the H⑀/C⑀ and H␥/C␥ atoms were used (16) Table S1). To avoid the peak broadening that characterizes NMR spectra of the 1:2 interaction, samples of the 1:1 complex between labeled CaM and unlabeled LSELl or LSEL15 were created through careful titration monitored through 15 N or 13 C HSQC spectra: 0.1 molar increments of peptide were added to CaM until the cross-peaks of free Ca 2ϩ -CaM were replaced with those of the 1:1 spectra. For resonance assignment of the bound peptide, a 0.7:1 molar ratio of peptide to CaM was employed to ensure peptide saturation.…”

“…The structure calculation of the Ca 2ϩ -CaM⅐LSEL15 complex was performed using a previously established two-step, low temperature torsion angle simulated annealing protocol in the program Xplor-NIH (version 2.24) (15,26,27). Briefly, in the first step, the starting model built from the homologous crystal structure underwent Powell energy minimization and torsion angle dynamics at 200 K followed by simulated annealing in which the temperature was decreased from 200 to 20 K in ⌬T ϭ 10 K steps.…”