Calcium-binding EF-hand proteins and protein domains; an NMR and biophysical approach

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Eukaryotic cells contain a remarkable variety of proteins that have to work together in a coordinated manner to achieve a specific physiological outcome. In order to attain such control, the activities of many proteins are regulated by various chemical signals, that can turn specific groups of proteins on and off. There are a number of distinct chemical signals that can orchestrate these cellular responses, such as cyclic nucleotides and calcium. The research program described here, is focussed on a group of regulatory calcium-binding proteins, called the EF-hand proteins. This name refers to a specific helix-loop-helix structural motif, that was found when the 3D structure of the first protein of this class was determined in the 1970's. By now we know that human and plant genomes code for over 250 distinct EF-hand proteins and that most of these have multiple EF-hand motifs. For example, the most widely distributed protein calmodulin, has four EF-hand motifs that are organized in a pairwise manner into two connected lobes. Calmodulin is present in all eukaryotes, ranging from humans, birds, fish, amoeba, plants and yeast, indicating that this is an almost universal signalling system. While calcium is the signal, the EF-hand proteins are the on- and off-switches: after binding calcium they can bind to and alter the activity of their target proteins. Since the 1980's the structures of many EF-hand proteins, or EF-hand domains that are part of larger proteins, have been solved, and their functions have been elucidated through elegant cell biology studies. It has recently become clear, however, that plants and yeasts contain a number of EF-hand proteins, that carry specific mutations, and these can no longer respond to calcium in the same manner. Nonetheless, these 'inactivated switches' are completely conserved, suggesting that they must have another important biological function; we will investigate these poorly understood functions for several plant and yeast actin-bundling fimbrin/plastin proteins, and in ATP-magnesium mitochondrial carrier proteins. We will also study another multi-domain EF-hand protein called nucleobindin2; this protein is unique as it is secreted into the Endoplasmic Reticulum, where the calcium regulation is very different from that in the cytoplasm. Nucleobindin2 provides the precursor for the peptide hormone nesfatin, and we plan to demonstrate that its EF-hand domain can play a decisive role in the proteolytic release of this hormone. Finally, we will investigate in detail the interactions between calmodulin and the aquaporin 0 and 4 transmembrane water channels, as this leads to intriguingly different physiological outcomes, channel inhibition in the former and channel translocation in the latter case. Taken together, these studies will provide new molecular level insights into the biological roles of these unique EF-hand protein interactions and further expand our understanding of their evolutionary relationships.