The molecular bases of many biological regulation processes are based on the precisely controlled use of protein/protein interactions. By altering these in response to various cues, changes can be produced in protein conformations (via in cis interactions) or the composition of multiprotein complexes (in trans interactions). Either type of these changes can in turn be harnessed to affect signaling through various intracellular pathways.
To achieve this in practice, two essential functions must be built into these molecular switches:
1). Sensory mechanisms to detect changes in various external stimuli
2). Transduction mechanisms to convert these signals into conformational changes that alter protein/protein interactions
Both of these are elegantly integrated within members of the PAS (Per-Arnt-Sim) family of protein/protein interaction domains. These small (~110 amino acid) modules are found in organisms distributed through all three kingdoms of life, where they serve as components of enzymes, transcription factors and ion channels. Within these proteins, PAS domains often play sensory roles by monitoring physical phenomena as diverse as redox state, oxygen concentration or light, depending on the specific domain. To respond to such wide-ranging stimuli, these domains must complement the chemical diversity of the twenty natural amino acids by binding various organic cofactors. Environmental changes alter the conformation or binding of these ligands, transforming the surrounding protein structure in ways that alter the domains ability to partake in various protein/protein interactions.
To gain a better understanding of the molecular basis of the sensory and regulatory mechanisms used by PAS domains, we are combining biophysical, biochemical and chemical approaches to study two classes of eukaryotic PAS-containing proteins:
Kinases: Two types of kinases are under investigation within our group, both of which are regulated by PAS domains in cis. The first of these, PAS kinase, is conserved from yeast to humans. The N-terminal PAS domain of this protein can specifically inhibit its own kinase, suggesting a direct interaction between these two. We have solved the solution structure of this domain and identified the region that is responsible for this interaction (Amezcua et al., 2002). To examine how this domain might be modulated by small organic cofactors, we have assembled a library of over 800 small organic chemicals and subsequently screened this to identify PAS-binding compounds. These results suggest that the ability to specifically bind ligands is widely spread through the PAS family. These compounds have also proven to be useful in generating artificial regulators of other protein/protein interactions as well (Best et al., 2004).
In parallel, we are also investigating the used within the phototropins, a family of light-activated plant kinases. These mediate several biological responses to blue light exposure, including phototropism, stomatal opening and chloroplast reorganization. Specialized flavin-binding PAS domains within these proteins serve as photosensors, converting blue light into a signal that activates kinase activity. Intriguingly, these domains are used by the bread mold Neurospora crassa to synchronize its circadian rhythms to the daily light/dark cycle (He et al., 2002). Our primary interest for these systems is characterize how light can be converted into structural and dynamic changes within the PAS domains to initiate these signaling pathways. Studies of one of these domains from the oat phototropin1 protein has recently shown that light perturbs an intramolecular association between the PAS domain and an additional alpha helix, constituting a protein-based switch that changes conformation in response to illumination (Harper et al., 2003; Harper et al., 2004).
Transcription factors: PAS domains are also found within a number of eukaryotic transcription factors, including members of the bHLH-PAS family (e.g. hypoxia inducible factor (HIF) (Erbel et al., 2003), NPAS2; Holdeman and Gardner, 2001) and the Neurospora white collar circadian clock system (He et al., 2003). In these proteins, PAS domains appear to be involved in the interactions that are needed to build heterodimeric complexes that can bind DNA and activate transcription. We are studying PAS domains from several of these factors, using NMR-based studies to investigate the structural basis of specificity in their protein/protein interactions. As we have found in the HIF/ARNT heterodimer (Erbel et al., 2003; Card et al. 2005), the intermolecular interactions utilize PAS domain surfaces that are analogous to those used for intramolecular interactions in the phototropin and PYP systems (Harper et al., 2003; Harper et al., 2004).
RESEARCH INTERESTS
biophysics
NMR
PAS domains
protein/protein interactions
ligand regulation
RECENT PUBLICATIONS
Card, P.B., Erbel, P.J.A. and Gardner, K.H., "Structural basis of ARNT PAS-B dimerization: Use of a common β-sheet interface for hetero- and homodimerization" J. Mol. Biol., 353:664-677, 2005
Socolich, M., Lockless, S.W., Russ, W.P., Lee, H., Gardner, K.H. and Ranganathan, R., "Evolutionary information for specifying a protein fold" Nature, 437:512-518, 2005
Yang, J., Zhang, L., Erbel, P.J.A., Gardner, K.H., Ding, K.M., Garcia, J.A. and Bruick, R.K., "Functions of the Per/ARNT/Sim (PAS) domains of the hypoxia inducible factor (HIF)" J. Biol. Chem., 280:36047-36054, 2005
Colbert, C.L., Wu, Q., Erbel, P.J.A., Gardner, K.H. and Deisenhofer, J., "Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation" Proc. Natl. Acad. Sci., 103:4410-4415, 2006
Kajuimura, J., Rahman, A., Hsu, J., Evans, M.R., Gardner, K.H. and Rick, P.D., "O-acetylation of enterobacterial common antigen (ECA) polysaccharide is catalyzed by the product of the yiaH gene of Escherichia coli K-12" J. Bacteriology, 188:7542-7550, 2006
SIGNIFICANT PUBLICATIONS
Amezcua, C.A., Harper, S.M., Rutter, J., and Gardner, K.H., "Structure and interactions of PAS kinase N-terminal PAS domain: Model for intramolecular kinase regulation" Structure, 10:1349-1361, 2002
Harper, S.M., Neil, L.C. and Gardner, K.H., "Structural basis of a phototropin light switch" Science, 301:1541-1544, September 2003
Erbel, P.J.A., Card, P.B., Karakuzu, O., Bruick, R.K. and Gardner, K.H., "Structural basis of PAS domain heterodimerization in the basic helix-loop-helix-PAS transcription factor hypoxia-inducible factor" Proc. Natl. Acad. Sci., 100:15504-15509, December 2003
Harper, S.M., Neil, L.C., Day, I.J., Hore, P.J. and Gardner, K.H., "Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy" J. Am. Chem. Soc., 126:3390-3391, 2004
Card, P.B., Erbel, P.J.A. and Gardner, K.H., "Structural basis of ARNT PAS-B dimerization: Use of a common β-sheet interface for hetero- and homodimerization" J. Mol. Biol., 353:664-677, 2005
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