A. The structural basis for selectivity in intracellular Ca2+ signal transduction.
The EF-hand is a very abundant protein motif, but despite its widespread distribution, very little is known about what properties provide the specific biochemical functions that enable participation in specific biological processes. The long-term objective of our research is to determine the underlying molecular basis for the specificity of intracellular Ca2+ signal transduction. The goals are to understand how the sequence of an EF-hand protein specifies its response to the binding of Ca2+, and how the differences in the structural and biochemical properties of Ca2+-activated EF-hand proteins enable interaction with and modulation of specific protein targets. This research is supported by NIH RO1 GM40120.
1. Engineering Ca2+-induced conformational change in EF-hand proteins by rational design. (Skelton 1993; Nelson 1998, 2002; Maler 2000; Bunick 2004)
The long-term goal in this project is to determine the relationship between sequence and function in EF-hand proteins, so that we can use this knowledge to manipulate Ca2+ signaling pathways in therapeutic and biotechnological settings. The approach involves an iterative rational protein engineering strategy designed to elucidate the factors controlling the structural response of EF-hand proteins to the binding of Ca2+. Two prototypical EF-hand proteins, calbindin D9k and calmodulin, are used as the framework for these studies because their biochemical properties and biological functions are vastly different. Single and limited-site mutations in calbindin D9k are made to establish the importance of specific residues or groups of residues in the protein sequence. The focal point of our efforts is the creation of a novel hybrid protein that we call calbindomodulin, i.e. calbindin D9k re-engineered to respond to the binding of Ca2+ in the manner of calmodulin.
2. The molecular basis for target activation by centrin. (Veeraraghavan 2002, Hu 2003)
This project addresses issues relating to the specificity of targeting by different EF-hand proteins. It involves study of centrin, which is a very close homolog of the ubiquitous Ca2+ sensor, calmodulin (CaM). Centrin is an essential component of the centrosome, which mediates chromosome segregation during mitosis. It has ~50% homology with CaM and a similar global architecture comprised of two EF-hand domains. However, several key characteristics distinguish centrin from CaM and there is strong evidence that they function by fundamentally different mechanisms. High resolution 3D structure is being used to establish how centrin's interactions with its targets differ from those of CaM. We have shown that unlike calmodulin, interaction with the yeast target Kar1p involves only centrin's C-terminal domain. The structure of the complex revealed several key features that distinguish centrin-Kar1p from CaM-target systems. The current emphasis in this project is to find N-terminal domain target(s) using yeast proteome chips or other approaches. The system(s) will be characterized biophysically and structurally, initially using the N-terminal domain alone and then the ternary complex, so that we can ultimately develop a full picture of centrin function.
3. The molecular basis for target activation by CDPK. (Christodoulou 2002,2004)
CDPK (calcium-dependent protein kinase) is a unique protein kinase found only in plants and protozoa including Plasmodium falciparum, the causative agent of malaria. CDPK is distinguished from all other kinases because it contains its own calmodulin-like regulatory apparatus in a domain extending from the C-terminus of the kinase catalytic domain. This unusual arrangement imposes a mechanism of kinase activation on CDPK that must be distinct from the current calmodulin-dependent kinase paradigm. Our objective in this project is to define the molecular basis for Ca2+-dependent inhibition/activation of CDPK's kinase activities using biophysical, structural and functional studies of CDPK's regulatory apparatus. In addition, 3D structural data and screening approaches are being used to develop selective CDPK inhibitors as a concept for anti-malarial therapeutics. Incorporation of negative design away from inhibition of the essential and ubiquitous activator calmodulin is a key aspect of these studies, as this design feature must be an essential part of all therapeutics directed to EF-hand proteins.
4. Ca2+ regulation of gating of the human cardiac Na+ channel. (Wingo 2004)
Recent studies in our laboratory have disclosed the presence of an EF-hand domain in the proximal C-terminal region of the human cardiac Na+ channel, hH1. Previous electrophysiological studies in the laboratory of my collaborator, Jeff Balser, demonstrated a Ca2+ effect on gating of hH1 that was originally assigned to Ca2+-dependent interactions between calmodulin and a putative IQ motif in the C-terminal region. However, a subsequent multiple sequence alignment revealed the presence of a single consensus EF-hand motif. Using a combination of sub-cloning and site directed mutagenesis coupled to electrophysiological, biophysical and structural modeling experiments, we obtained clear evidence that a fully functional EF-hand domain exists in the proximal C-terminal region of hH1. Moreover, several mutations shown to lead to long QT and Brugada syndromes map to the EF-hand Ca2+ binding loop and we showed that these mutations are defective in Ca2+ binding. Current efforts are focused on structure determination of the EF-hand domain to facilitate evaluating the potential of targeting this site for therapy in the treatment of cardiac arrhythmia syndromes.
last updated April 26, 2004