Johan Evenäs

Calcium-induced structural changes and domain autonomy in calmodulin.

We have determined the solution structures of the apo and (Ca2+)2 forms of the carboxy-terminal domain of calmodulin using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The results show that both forms adopt well-defined structures with essentially equal secondary structure. A comparison of the structures of the two forms shows that Ca2+ binding causes major rearrangements of the secondary structure elements with changes in inter-residue distances of up to 15 Å and exposure of the hydrophobic interior of the four-helix bundle. Comparisons with previously determined high-resolution X-ray structures and models of calmodulin indicate that this domain is structurally autonomous.

Dynamics of the Transition between Open and Closed Conformations in a Calmodulin C-Terminal Domain Mutant

BACKGROUND Calmodulin is a ubiquitous Ca(2+)-activated regulator of cellular processes in eukaryotes. The structures of the Ca(2+)-free (apo) and Ca(2+)-loaded states of calmodulin have revealed that Ca(2+) binding is associated with a transition in each of the two domains from a closed to an open conformation that is central to target recognition. However, little is known about the dynamics of this conformational switch. RESULTS The dynamics of the transition between closed and open conformations in the Ca(2+)-loaded state of the E140Q mutant of the calmodulin C-terminal domain were characterized under equilibrium conditions. The exchange time constants (tau(ex)) measured for 42 residues range from 13 to 46 micros, with a mean of 21 +/- 3 micros. The results suggest that tau(ex) varies significantly between different groups of residues and that residues with similar values exhibit spatial proximity in the structures of apo and/or Ca(2+)-saturated wild-type calmodulin. Using data for one of these groups, we obtained an open population of p(o) = 0.50 +/- 0.17 and a closed --> open rate constant of k(o) = x 10(4) s(-1). CONCLUSIONS The conformational exchange dynamics appear to involve locally collective processes that depend on the structural topology. Comparisons with previous results indicate that similar processes occur in the wild-type protein. The measured rates match the estimated Ca(2+) off rate, suggesting that Ca(2+) release may be gated by the conformational dynamics. Structural interpretation of estimated chemical shifts suggests a mechanism for ion release.

When Size Is Important ACCOMMODATION OF MAGNESIUM IN A CALCIUM BINDING REGULATORY DOMAIN

The accommodation of Mg2+ in the N-terminal domain of calmodulin was followed through amide1H and 15N chemical shifts and line widths in heteronuclear single-quantum coherence spectroscopy NMR spectra. Mg2+ binds sequentially to the two Ca2+-binding loops in this domain, with affinities such that nearly half of the loops would be occupied by Mg2+ in resting eukaryotic cells. Mg2+ binding seems to occur without ligation to the residue in the 12th loop position, previously proven largely responsible for the major rearrangements induced by binding of the larger Ca2+. Consequently, smaller Mg2+-induced structural changes are indicated throughout the protein. The two Ca2+-binding loops have different Mg2+ binding characteristics. Ligands in the N-terminal loop I are better positioned for cation binding, resulting in higher affinity and slower binding kinetics compared with the C-terminal loop II (k off = 380 ± 40 s–1compared with ∼10,000 s−1 at 25 °C). The Mg2+-saturated loop II undergoes conformational exchange on the 100-μs time scale. Available data suggest that this exchange occurs between a conformation providing a ligand geometry optimized for Mg2+ binding and a conformation more similar to that of the empty loop.

Structural Insights into the Calcium-Mediated Allosteric Transition in the C-Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements

Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca(2+)-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, (15)N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca(2+) by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca(2+)-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations.

1H, 15N and 13C assignments and secondary structure of the EGF-like module pair 3–4 from vitamin K-dependent protein S

Vitamin K‐dependent protein S, which is a cofactor for activated protein C and thus important for down‐regulation of the coagulation cascade, contains several Ca2+‐binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor‐like (EGF) modules of protein S is the smallest fragment that retains high‐affinity Ca2+ binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the 1H, 15N and 13C resonances of the module pair with one Ca2+ bound in EGF 4. In addition, nearly complete assignments of the 1H resonances of the isolated Ca2+‐free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis‐trans isomerisation of the peptide bond preceding Pro‐168, and dimerisation. Analysis of weighted average secondary chemical shifts, 3 J HNHα coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti‐parallel β‐sheet.