Experimentally exploring the conformational space sampled by domain reorientation in calmodulin

Abstract

The conformational space sampled by the two-domain protein calmodulin has been explored by an approach based on four sets of NMR observables obtained on Tb 3؉ -and Tm 3؉ -substituted proteins. The observables are the pseudocontact shifts and residual dipolar couplings of the C-terminal domain when lanthanide substitution is at the N-terminal domain. Each set of observables provides independent information on the conformations experienced by the molecule. It is found that not all sterically allowed conformations are equally populated. Taking the N-terminal domain as the reference, the C-terminal domain preferentially resides in a region of space inscribed in a wide elliptical cone. The axis of the cone is tilted by Ϸ30°with respect to the direction of the N-terminal part of the interdomain helix, which is known to have a flexible central part in solution. The C-terminal domain also undergoes rotation about the axis defined by the C-terminal part of the interdomain helix. Neither the extended helix conformation initially observed in the solid state for free calcium calmodulin nor the closed conformation(s) adopted by calcium calmodulin either alone or in its adduct(s) with target peptide(s) is among the most preferred ones. These findings are unique, both in terms of structural information obtained on a biomolecule that samples multiple conformations and in terms of the approach developed to achieve the results. The same approach is in principle applicable to other multidomain proteins, as well as to multiple interaction modes between two macromolecular partners. C almodulin (CaM) is a paradigm case in structural biology. The following brief survey of the history of the structural and dynamic studies on this protein serves the double purpose of putting the present findings in proper perspective and of acknowledging those pieces of previous information that were essential to allow the present approach to be developed and to yield novel structural information.CaMs are two-domain proteins belonging to the large family of EF-hand proteins (1-3). They contain Ϸ150 amino acid residues, organized into two domains of Ϸ70 aa each and connected by a short linker. Each domain is made up of two special helix-loop-helix motifs (EF-hand motifs) that can bind a calcium ion in the loop. The two loops are held close to one another by two short antiparallel ␤-strands forming a threehydrogen bond stretch of ␤-sheet. The function of CaM in cell cytoplasm is that of responding to sudden rises of calcium concentration by binding up to four calcium ions in the four EF-hand loops, by changing conformation because of metal binding, and by thus becoming able to recognize, bind to, and activate, a number of proteins and enzymes (1,(4)(5)(6)(7)(8). Early x-ray data (9) showed the four-calcium (Ca 2 ) N (Ca 2 ) C CaM form (subscripts N and C refer to the calcium atoms bound by the N-and C-terminal domains, respectively) to have a dumbbell shape, with helix 4, the last helix of the N-terminal domain, and helix 5, the first helix of the C-termi...

Protein-Protein Docking and Analysis Reveal That Two Homologous Bacterial Adenylyl Cyclase Toxins Interact with Calmodulin Differently

“…We ran the docking with and without the following restraints: the distance restraints were placed to move the second calcium-binding, EF-hand motif close to the ␤-hairpin of CyaA-ACD. A restraint was also placed to anchor the C-terminal end of N-CaM to the N terminus of C-CaM, because N-CaM is linked to C-CaM by a flexible linker with a defined degree of freedom (9). Unfortunately, no satisfactory model emerged from our docking analysis.…”

“…However, C-CaM alone cannot activate CyaA as potently as full-length CaM. Based on the fact that Nand C-CaM are joined by a flexible linker, which enables them to bind to two distinct sites of a target molecule cooperatively, we placed the N-terminal calcium-free closed conformations of calmodulin models from NMR structures (PDB code 1CFC) into the C-CaM-bound CyaA-ACD structure model (PDB code 1YRT) (3,9). We found several models that did not physically crash with the C-CaM bound CyaA-ACD structure and could have an acceptable match between the shapes and electrostatic potential energy surfaces of both proteins.…”

“…The molecular structures of Ca 2ϩ -bound and Ca 2ϩ -free CaM have been determined by x-ray crystallography and NMR (4 -8). CaM has two globular domains, termed N-CaM and C-CaM, which are connected by a flexible, central ␣-helix (9). Each domain is composed of two helix-loop-helix EF-hand calcium binding motifs (4).…”

Paramagnetic shifts in solid-state NMR of proteins to elicit structural information

“…However, even when additional dihedral angle restraints from backbone chemical shiftsthrough Chemical Shift Index (CSI) (19) or TALOS (20) programs-are used, the size of the affordable proteins has been, up to now, limited to small systems (Ͻ100 aa) (10,15,18). In this work, we show how SSNMR paramagnetic restraints such as pseudocontact shifts (pcs) can be used as additional sources of restraints for protein structural determination, even providing information about the relative arrangement of protein molecules in the solid phase.Paramagnetic NMR restraints as relaxation times, pcs, and residual dipolar couplings (RDC) (21)-the latter two originating from anisotropy in the magnetic susceptibility tensor-are routinely used in solution NMR to refine structures (22), to investigate protein-protein interactions (23, 24), or to monitor dynamics (25,26). Small paramagnetic molecules have been studied through magic angle spinning (MAS) SSNMR for decades (27-33).…”

“…Paramagnetic NMR restraints as relaxation times, pcs, and residual dipolar couplings (RDC) (21)-the latter two originating from anisotropy in the magnetic susceptibility tensor-are routinely used in solution NMR to refine structures (22), to investigate protein-protein interactions (23, 24), or to monitor dynamics (25,26). Small paramagnetic molecules have been studied through magic angle spinning (MAS) SSNMR for decades (27-33).…”