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Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3.

ABSTRACT: Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.

SUBMITTER: Drake SK

PROVIDER: S-EPMC2233790 | biostudies-literature | 1997 Aug

REPOSITORIES: biostudies-literature

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Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3.

Drake S K SK Zimmer M A MA Kundrot C C Falke J J JJ

The Journal of general physiology 19970801 2

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of ...[more]

PMID: 9236210

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Project description:The EF hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca(2+) and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca(2+) regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-hand domain serve as coupled Ca(2+) sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to define their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.

Dataset Information

Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3.

Publications

Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3.

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