Department of Biochemistry and Molecular Biology
RBHS, Robert Wood Johnson Medical School

Cancer Institute of New Jersey

Ph.D., 1986, University of California, Berkeley
Telephone: (848) 445-9812
Fax: (732) 235-5289

Structure/function analysis of bacterial signal transduction pathways

Research in the Stock laboratory focuses on understanding the structure and function of signal transduction proteins and in particular, how covalent modifications regulate protein activities. All cells monitor their surrounding environments and elicit appropriate adaptive 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.

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 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. A combination of biophysical and biochemical approaches are used in the Stock laboratory to investigate how these molecular switch proteins function to 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 subclassified 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, with multiple family members encoded in a single genome and >5000 different OmpR/PhoB proteins identified to date. This large family allows investigation of a basic question of broad relevance. Do homologous signaling proteins with structurally similar domains use common mechanisms to regulate function?

The short answer to this question is "no". For all two-component proteins, there is a limit to the extent that sequence and structural similarity can be used to predict mechanisms of function. Properties intrinsic to individual domains are conserved while domain arrangements and corresponding modes of regulation are diverse. Mechanisms of regulation in the OmpR/PhoB family are complex, displaying 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. Recent studies indicate that OmpR/PhoB family members have different inactive states but adopt a common active state upon phosphorylation (see figure). Structural and biochemical analyses indicate that different domain arrangements provide diverse 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 competent for phosphorylation.

Additional projects in the laboratory focus on characterization of two-component signaling pathways in Staphylococcus aureus. Drug resistant Staph (e.g. methicillin-resistant S. aureus, MRSA) is an emerging health problem. Indeed, annual deaths in the US involving S. aureus currently outnumber those involving HIV. Two-component systems mediate processes fundamental to Staph pathogenesis including regulation of virulence factor expression and adaptation to antibiotics. Structure/function studies are aimed at defining the specific roles of two-component proteins and in identifying inhibitors of these novel therapeutic targets.

Figure 1. Inactive and active domain arrangements in the OmpR/PhoB family. Although OmpR/PhoB response regulators have similar domain architectures, arrangements of the domains in the inactive state are diverse. Upon activation by phosphorylation, response regulators adopt a common dimeric structure. Inactive response regulators are shown aligned relative to the regulatory domains (blue, α4-β5-α5 face gold), illustrating the different arrangements of the DNA-binding domains (green, recognition helix red).

Selected Publications

Gao R, Godfrey KA, Sufian MA, Stock AM. (2017) Counterbalancing regulation in response memory of a positively autoregulated two-component system. J Bacteriol 199:e00390-17

Gao R, Stock AM. (2017) Quantitative kinetic analyses of shutting off a two-component system. mBio 8:e00412-17

Forest KT, Stock, AM. (2016) Classic spotlight: crowd sourcing provided Penicillium strains for the war effort. J Bacteriol 198:877

Gao R, Stock, AM. (2015) Temporal hierarchy of gene expression mediated by transcription factor binding affinity and activation dynamics. MBio 6:e00686-15

Winkelmann DA, Forgacs E, Miller MT, Stock AM. (2015) Structural basis for drug-induced allosteric changes to human beta-cardiac myosin motor activity. Nat Commun 6:7974

Gao R, Stock AM. (2013) Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling. Proc Natl Acad Sci USA 110:672-7

Leonard PG, Golemi-Kotra D, Stock AM. (2013) Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation. Proc Natl Acad Sci USA 110:8525-30

Gao R, Stock AM. (2013) Evolutionary tuning of protein expression levels of a positively autoregulated two-component system. PLoS Genet 9:e1003927

Gao R, Stock AM. (2009) Biological insights from structures of two-component proteins. Annu Rev Microbiol 63:133-54

Sidote DJ, Barbieri CM, Wu T, Stock AM. (2008) Structure of the Staphylococcus aureus AgrA LytTR domain bound to DNA reveals a beta fold with an unusual mode of binding. Structure 16:727-35