All 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, which regulates the activities of proteins. We are investigating this signaling mechanism from both structural and functional perspectives.
Two-Component Signal Transduction in Bacteria
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 antimicrobial 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 conserved regulatory domain and a variable 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 their associated effector domains to elicit specific responses such as flagellar rotation, regulation of transcription, or enzymatic catalysis. We use a combination of biophysical and biochemical approaches to investigate how these molecular-switch proteins regulate cellular activities.
The majority of bacterial response regulators are transcription factors that regulate expression of specific sets of genes in response to 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. More than 3,000 different OmpR/PhoB family proteins have been identified. This large family allows us to pose a 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 limit to the extent that sequence and structural similarity can be used to predict mechanisms of function. The mechanisms of regulation in the OmpR/PhoB family are complex, however, displaying both similarities and differences. In well-characterized members of this 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 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 two-component signaling system.
We have determined the structures of inactive conformations of four 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. 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 intramolecular interactions, and modulation of the phosphorylation rate 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 α4-β5-α5 face of the domain, the region that differs 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 (Figure 1). Dimerization of the regulatory domains brings the DNA-binding domains into close proximity, promoting binding to tandemly arrayed DNA half-sites. The conserved dimerization surface used by OmpR/PhoB family members suggested that these proteins could form heterodimers. Indeed, we have demonstrated that heterodimers between different OmpR/PhoB family members form readily in vitro. We are currently probing the physiological significance of heterodimer formation in vivo. Formation of heterodimers would provide an elegant mechanism for integrating signals from different signaling pathways. Thus the large OmpR/PhoB family may provide a network for coordinating adaptive responses to complex signals within bacterial cells. (Our investigations of response regulator proteins are supported in part by 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 (Robert Wood Johnson Medical School), to be the molecular locus of Niemann-Pick type C2 disease. This fatal hereditary disorder is characterized by accumulation of low-density lipoprotein–derived cholesterol in lysosomes, resulting in progressive neurodegeneration. The disease has provided significant clues to the proteins involved in trafficking cholesterol inside cells, a process that involves the membrane transporter NPC1 and the soluble protein NPC2, which together appear to facilitate egress of cholesterol from lysosomes.
In collaboration with the Lobel laboratory, we have pursued structural and biochemical characterization of the NPC2 protein, which binds cholesterol and a variety of different sterols with submicromolar affinity. The x-ray crystal structure has revealed that NPC2 has a simple β-sandwich fold (Figure 2), but unlike most other lipid- and sterol-binding proteins, lacks a preformed hydrophobic cavity for ligand binding. The binding cavity is formed only in the presence of the ligand, by a slight separation of a few strands of the two β sheets and the reorientation of several side chains. The hydrophobic portion of the sterol is inserted between the β sheets, and the protein molds itself around the ligand, creating a perfectly fitting tunnel that penetrates deep into the interior of the protein. The ligand-binding site thus formed is highly malleable, as evidenced by the tolerance of sterol-binding function to variations in both the protein and the ligand. It is likely that the physiologically relevant ligands bound by NPC2 are dictated by the specific subcellular repertoire of sterols available for binding, rather than by stringent selectivity of the binding cavity. (Structural studies of NPC2 are funded in part by the Ara Parseghian Medical Research Foundation.)
