Abstract
Binding of calcium to calmodulin (CaM) causes a conformational change in this ubiquitous calcium regulatory protein that allows the activation of many target proteins. Met residues make up a large portion of its hydrophobic target binding surfaces. In this work, we have studied the surface exposure of the Met residues in the apo- and calcium-bound states of CaM in solution. Complexes of calcium-CaM with synthetic peptides derived from the CaM-binding domains of myosin light chain kinase, constitutive nitric-oxide synthase, and CaM-dependent protein kinase I were also studied. The surface exposure was measured by NMR by studying the effects of the soluble nitroxide spin label, 4-hydroxyl-2,2,6, 6-tetramethylpiperidinyl-1-oxy, on the line widths and relaxation rates of the Met methyl resonances in samples of biosynthetically 13C-methyl-Met-labeled CaM. The Met residues move from an almost completely buried state in apo-CaM to an essentially fully exposed state in Ca2+4-CaM. Binding of two Ca2+ to the C-terminal lobe of CaM causes full exposure of the C-terminal Met residues and a partial exposure of the N-terminal Met side chains. Binding of the three target peptides blocks the access of the nitroxide surface probe to nearly all Met residues, although the mode of binding is distinct for the three peptides studied. These data show that calcium binding to CaM controls the surface exposure of the Met residues, thereby providing the switch for target protein binding.
Highlights
Calmodulin (CaM)1 [1] is a ubiquitous calcium regulatory protein that is found in all eukaryotic cells
We did not observe any changes in the EPR spectra recorded with TEMPOL/Ca2ϩ4-CaM at a ratio of 6, TEMPOL/apo-CaM at a ratio of 1⁄6 or 6, and the TEMPOL/Ca2ϩ4-CaM-myosin light chain kinases (MLCK)-peptide complex at a ratio of 1⁄6 or 6. The outcome of these EPR studies indicates that there are no specific interactions between TEMPOL and CaM; the soluble spin label is suitable to probe the exposed hydrophobic surfaces and the exposure of Met methyl groups in CaM [29, 32]
We have studied the surface exposure of the Met methyl groups of CaM in different physiologically relevant states by using the soluble spin label TEMPOL
Summary
(Received for publication, November 6, 1998, and in revised form, January 6, 1999). Tao Yuan‡, Hui Ouyang‡, and Hans J. Structural and dynamic information derived by NMR spectroscopy as well as molecular dynamics simulations indicates that the region between amino acids 77 and 81 in Ca2ϩ4-CaM is flexible in solution [9, 10] This central linker region in Ca2ϩ4-CaM unravels further when a CaM-binding peptide from a target protein such as MLCK is bound [11, 12]. 4-Hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPOL; called HyTEMPO) is a nonperturbing nitroxide-soluble spin label that can be used for mapping surface-exposed hydrophobic side chains in proteins It has been used for the characterization of different protein folding intermediates, identification of the critical residues involved in ligand-binding, and simplification of 1H NMR spectra for assignment purposes Our results reveal that changes in the surface exposure of the Met side chains play a key role in allowing CaM to act as a versatile calcium regulatory protein
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