Bryan E. Finn

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.

The evolving model of calmodulin structure,function and activation

Recent high-resolution crystal and solution structures have answered many long-standing questions about calmodulin and its various conformational states. However, there is still much to learn.

Dissection of Calbindin D9k into two Ca2+-binding subdomains by a combination of mutagenesis and chemical cleavage

Calbindin D9k is a 75‐residue globular protein made up of two Ca2+‐binding subdomains of the EF‐hand type. In order to examine the subdomains independently, a method was devised to selectively cleave the loop between them. Using site‐directed mutagenesis, a unique methionine was substituted for Pro43 in the loop, thus allowing cleavage using cyanogen bromide. Agarose gel electrophoresis shows that the fragments have a high affinity for one another, although less so in the absence of calcium.1H‐NMR spectra of the fragments indicate that the structures of the heterodimers are changed little from that of the intact protein. However, the Ca2+ binding constants of the individual subdomains are several orders of magnitude lower than for the corresponding sites in the uncleaved protein.

The structure of apo-calmodulin. A 1H NMR examination of the carboxy-terminal domain.

The structure of the carboxy-terminal domain of bovine calmodulin, TR2C, in the calcium-free form was investigated using two-dimensional 1H NMR. Sequential resonance assignments were made using standard methods. Using information from medium and long range contacts revealed by nuclear Overhauser enhancement, the secondary structure and global fold were determined. The apo protein possesses essentially the same secondary structure as that in the calcium activated form of intact calmodulin. However, the secondary structural elements are rearranged so that the hydrophobic binding pocket is closed in the apo-form.The structure of the carboxy‐terminal domain of bovine calmodulin, TR2C, in the calcium‐free form was investigated using two‐dimensional 1H NMR. Sequential resonance assignments were made using standard methods. Using information from medium and long range contacts revealed by nuclear Overhauser enhancement, the secondary structure and global fold were determined. The apo protein possesses essentially the same secondary structure as that in the calcium activated form of intact calmodulin. However, the secondary structural elements are rearranged so that the hydrophobic binding pocket is closed in the apo‐form.

Coupling of ligand binding and dimerization of helix-loop-helix peptides: Spectroscopic and sedimentation analyses of calbindin D9k EF-hands

Isolated Ca2+‐binding EF‐hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+‐binding and peptide association for two EF‐hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF‐hand fragments from calbindin D9k. A series of Ca2+‐titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF‐hands. The fragment containing the N‐terminal EF‐hand shows a strong tendency to dimerize in the Ca2+‐bound state. The average Ca2+‐affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+‐free monomer to fully loaded dimer, showing that a Ca2+‐free EF‐hand folds upon dimerization to a Ca2+‐bound EF‐hand, thereby presenting a preformed binding site to the second Ca2+‐ion. The C‐terminal EF‐hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF‐hand fragments are similar and in the range lgK = 4.6–5.3. Proteins 2002;47:323–333.

A MODEL FOR TARGET PROTEIN BINDING TO CALCIUM-ACTIVATED S100 DIMERS

S100 proteins are a family of dimeric calcium‐binding proteins implicated in several cancers and neurological diseases. Calbindin D9k is an unusual monomeric member of the S100 family. A calbindin D9k mutant containing a novel calcium‐induced helix is characterized. Based on sequence comparison, this helix could be a component of other S100 proteins and a factor in target protein binding. The origin of structural differences between three reported apo S100 dimer structures is verified. We conclude that the differences are a result of modeling rather than a function of different target binding properties. A mechanism for target protein binding is suggested.

Research lettersThe structure of apo-calmodulin: A 1H NMR examination of the carboxy-terminal domain

The structure of the carboxy-terminal domain of bovine calmodulin, TR2C, in the calcium-free form was investigated using two-dimensional 1H NMR. Sequential resonance assignments were made using standard methods. Using information from medium and long range contacts revealed by nuclear Overhauser enhancement, the secondary structure and global fold were determined. The apo protein possesses essentially the same secondary structure as that in the calcium activated form of intact calmodulin. However, the secondary structural elements are rearranged so that the hydrophobic binding pocket is closed in the apo-form.The structure of the carboxy‐terminal domain of bovine calmodulin, TR2C, in the calcium‐free form was investigated using two‐dimensional 1H NMR. Sequential resonance assignments were made using standard methods. Using information from medium and long range contacts revealed by nuclear Overhauser enhancement, the secondary structure and global fold were determined. The apo protein possesses essentially the same secondary structure as that in the calcium activated form of intact calmodulin. However, the secondary structural elements are rearranged so that the hydrophobic binding pocket is closed in the apo‐form.

Calcium-Binding Proteins

Publisher Summary This chapter describes calcium-binding proteins. Calcium is of widespread and fundamental importance in biochemistry because the calcium ion functions as a second messenger—that is, one whose signals are propagated by proteins specifically evolved for this purpose. The chapter divides the proteins (or the protein domains) into: (1) intracellular, (2) calcium mediated membrane bound, and (3) extracellular. The EF hand is by far the most common motif for intracellular calcium- binding proteins. It is an approximately 30 amino acid long peptide chain composed of a central calcium-binding loop flanked by two alpha helixes. Both nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography played key roles in understanding the target binding of calmodulin. The functional role and interactions with target proteins remain an area of intense study. One target protein similar to calcyclin has been identified and is expressed predominantly in the brain.