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Ann Stock

Research Description

Research in our laboratory is focused on understanding the molecular mechanism of receptor-mediated signal transduction. All living cells monitor their surrounding environments and elicit appropriate responses to changing conditions. Such stimulus-response coupling is essential for numerous and diverse processes such as growth and development, metabolic regulation, and sensing. Signal transduction pathways, through which information is passed sequentially from one protein component to the next, provide the molecular mechanism for linking input signals to output responses. Despite great diversity in the types of stimuli and responses involved in different pathways, a limited number of fundamental molecular strategies are used for signal transduction. One such strategy is reversible covalent modification, a process that regulates the activities of proteins. Our laboratory is investigating this signaling mechanism from both structural and functional perspectives.

Two-Component Signal Transduction in Bacteria. Although signal transduction is most commonly studied in higher organisms, it is both prevalent and relevant in microorganisms. The ability to respond to environmental changes is essential for single-celled organisms to survive and thrive. Because adaptive responses are essential for general metabolic functions as well as for host-pathogen interactions, signal transduction proteins are key targets for development of anti-microbial drugs.

The majority of signal transduction in bacteria occurs through pathways known as "two-component" systems. These systems utilize a common mechanism involving transfer of a high-energy phosphoryl group from a histidine protein kinase to an aspartate residue of a response regulator protein. Response regulator proteins typically contain two domains: a regulatory domain and an effector domain. The regulatory domains of response regulator proteins can be thought of as phosphorylation-activated switches that are turned on and off by phosphorylation and dephosphorylation. In the phosphorylated state, the conserved regulatory domains activate effector domains to elicit specific responses such as flagellar rotation, regulation of transcription, or enzymatic catalysis. Using a combination of biophysical and biochemical approaches, our laboratory is investigating how these molecular switch proteins function to regulate cellular activities.

The majority of bacterial response regulators are transcription factors that activate or repress expression of specific sets of genes in response to chemical and/or physical environmental cues. Response regulator transcription factors can be classed into subfamilies based on structural similarity within their DNA-binding effector domains. The OmpR/PhoB subfamily, characterized by a winged-helix DNA-binding domain, is the largest subfamily and accounts for approximately one third of all response regulators. The genome of a single bacterium typically encodes 5-40 different OmpR/PhoB family transcription factors. Over 1000 different OmpR/PhoB family proteins have been identified to date and the number continues to increase exponentially along with the number of sequenced bacterial genomes. This large family of signaling proteins allows us to pose a very basic question of broad relevance. Do homologous signaling proteins with structurally similar domains use common mechanisms to regulate function?
Within the OmpR/PhoB family of response regulators, the short answer to this question is "no". There is a significant limit to the extent that sequence and structural similarity can be used to predict mechanisms of function. However, the mechanisms of regulation in the OmpR/PhoB family are complex, displaying an interesting set of both similarities and differences. In well-characterized members of the OmpR/PhoB family, phosphorylation-mediated activation involves a transition from inactive monomers to active dimers (and/or higher order oligomers) and this dimerization promotes DNA binding to direct repeat half-sites located within the promoters of regulated genes. Our recent studies, described below, indicate that OmpR/PhoB family members have different inactive states but adopt a common active state upon phosphorylation. The different inactive conformations provide the basis for different regulatory strategies that are optimized for the specific needs of each individual two-component signaling system.

There are a relatively small number of structures of OmpR/PhoB family members in their inactive states. We have determined four such structures of OmpR/PhoB family members from Escherichia coli , Mycobacterium tuberculosis , and Thermotoga maritima . Although the regulatory and DNA-binding domains within the different proteins all have similar folds, the domain orientations differ significantly in the four structures. The variations include tight regulatory/effector domain interfaces, lack of an interdomain interface, different relative orientations of the two domains, and a regulatory domain dimer that is distinct to the inactive state. Structural and biochemical analyses indicate that these different structures provide for a variety of different regulatory strategies including steric occlusion of the recognition helix that is required for DNA binding, competitive inhibition of the active state by alternative inter- and intra-molecular interactions, and modulation of the rate of phosphorylation by trapping the regulatory domain in an inactive conformation that is not amenable to phosphorylation.

We have determined the X-ray crystal structures of activated regulatory domains of eight different OmpR/PhoB family members. All have remarkably similar structures, consisting of a rotationally symmetric dimer mediated by the a 4- b 5- a 5 face of the domain, the region that is known to differ most between inactive and active states. Amino acid residues that form the dimerization interface are highly and exclusively conserved within the OmpR/PhoB subfamily, supporting the hypothesis that almost all family members adopt a common active state upon phosphorylation. From work in several laboratories, including our own, it is known that the DNA-binding domains of OmpR/PhoB family members bind to tandem DNA half-sites as head-to-tail dimers, distinct from the head-to-head symmetry of the regulatory domains. Thus we postulate that within active OmpR/PhoB dimers, flexible interdomain linkers allow different dimer symmetries in the regulatory and effector domains. This flexibility has so far precluded direct structural analysis of full-length proteins in their active states and our model is derived from characterization of isolated regulatory and effector domains. A common active state is unique to the OmpR/PhoB subfamily of response regulators and investigations of its significance are underway. (Our investigations of response regulator proteins are supported in part by a MERIT award from the National Institutes of Health.)

Niemann-Pick C2 Protein. An additional project in our laboratory focuses on NPC2, a protein that has been shown by our colleague, Peter Lobel (Department of Pharmacology, Robert Wood Johnson Medical School), to be the molecular locus of Niemann-Pick type C2 disease. NPC disease is a hereditary disorder characterized by accumulation of low-density lipoprotein-derived cholesterol in lysosomes, resulting in progressive neurodegeneration and early childhood death. The disease has provided significant clues to the proteins involved in trafficking cholesterol inside cells, a process about which relatively little is known. In collaboration with the Lobel laboratory, we have pursued structural and biochemical characterization of the NPC2 protein. We have recently determined the X-ray crystal structure of bovine NPC2 bound to cholesterol sulfate. This structure defines the cholesterol-binding pocket in NPC2. Comparison with our previously determined structure of NPC2 in the absence of sterol confirms our hypothesis that NPC2 represents an unusual class of lipid-binding proteins that lack a pre-formed binding pocket. In the presence of sterol, a loosely packed region of the hydrophobic core of the protein rearranges itself around the ligand, creating a hand hand-in-glove fit. This binding mode is consistent with our biochemical analyses that indicate that a variety of cholesterol analogs of different sizes and shapes can bind to NPC2. Additional investigations are aimed at identifying cellular partners that interact with NPC2 to facilitate intracellular trafficking of cholesterol. (Structural studies of NPC2 are funded in part by the Ara Parseghian Medical Research Foundation.)