We have studied the solution structure of skeletal muscle troponin C complexed with troponin I in the presence of calcium using small-angle X-ray and neutron scattering. 4Ca2+.troponin C in the complex has an extended dumbbell shape with a radius of gyration of 23.9 +/- 0.5 A and a maximum linear dimension of approximately 72 A, similar to the values obtained from the crystal structure coordinates of troponin C (Herzberg & James, 1985). Troponin I is even more extended than troponin C with a radius of gyration of 41 +/- 2 A and a maximum linear dimension of approximately 118 A. The centers-of-mass for each component of the complex are approximately coincident (< 10-A separation) as are their long axes, and the troponin I component encompasses the 4Ca2+.troponin C. These data provide new insights into the nature of the conformational arrangement of this important Ca(2+)-sensitive molecular switch.

Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca2+, the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, Rg, for the complex is approximately 20% smaller than that of uncomplexed Ca2+.calmodulin (16 vs 21 A), and the maximum dimension, dmax, for the complex is also about 20% smaller (49 vs 67 A). The peptide-induced conformational rearrangement of calmodulin is [Ca2+] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca2+.calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbell-shaped Ca2+.calmodulin. The solvent contrast dependence of Rg for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase.

Calmodulin (CaM) is the major intracellular receptor for Ca2+ and is responsible for the Ca2+-dependent regulation of a wide variety of cellular processes via interactions with a diverse array of target enzymes. Our current view of the structural basis for CaM enzyme activation is based on biophysical studies of CaM complexed with small peptides that model CaM-binding domains. A major concern with interpreting data from these structures in terms of target enzyme activation mechanisms is that the larger enzyme structure might be expected to impose constraints on CaM binding. Full understanding of the molecular mechanism for CaM-dependent enzyme activation requires additional structural information on the interaction of CaM with functional enzymes. We have utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first structural view of CaM complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). Our data show that CaM undergoes an unhindered conformational collapse upon binding MLCK and activates the enzyme by inducing a significant movement of the kinase's CaM binding and autoinhibitory sequences away from the surface of the catalytic core.

Small-angle X-ray and neutron scattering have been used to study the solution structures of calmodulin complexed with synthetic peptides corresponding to residues 342-366 and 301-326, designated PhK5 and PhK13, respectively, in the regulatory domain of the catalytic subunit of skeletal muscle phosphorylase kinase. The scattering data show that binding of PhK5 to calmodulin induces a dramatic contraction of calmodulin, similar to that previously observed when calmodulin is complexed with the calmodulin-binding domain peptide from rabbit skeletal muscle myosin light chain kinase. In contrast, calmodulin remains extended upon binding PhK13. In the presence of both peptides, calmodulin also remains extended. Apparently, the presence of PhK13 inhibits calmodulin from undergoing the PhK5-induced contraction. These data indicate that there is a fundamentally different type of calmodulin-target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase.

No abstract

We have successfully substituted 240 Pu 3+ for Ca 2+ in the calcium-binding protein calmodulin and used neutron resonance scattering from the bound 240 Pu to demonstrate that the Pu binds specifically to the Ca 2+ sites and also to measure the distance between the ion binding sites within individual domains of the protein. 240 Pu has a strong nuclear resonance at 0.278 Å, and at this wavelength the coherent scattering from 240 Pu is >1000 times that of any other nucleus present in a protein. The ionic radius of Pu 3+ is very similar to that of Ca 2+ , and hence we chose this species to substitute for Ca 2+ in the protein. We identified solution conditions that stablize Pu 3+ in solution at near neutral pH for 6-7 h in order to form the Pu/calmodulin complex under conditions favorable for both complex formation and maintaining the structural integrity of the protein. We collected small-angle neutron scattering data from solutions of 4( 240 Pu 3+ )‚calmodulin, which contain periodic terms that are directly related to the distances between the Ca 2+ -binding sites. The shorter Pu-Pu distance, i.e., the average distance between the two sites within each globular domain of calmodulin, is found to be 11.8 ( 0.4 Å, in excellent agreement with the value of 11.7 Å from crystallographic determinations. This is the first use of neutron resonance scattering as a structural probe in a protein.