Cellular signaling and homeostasis clearly rely on complex networks in which various input signals produce distinct outputs. This reliance is especially apparent in pathways responsible for normal growth and cell differentiation and the development of multicellular organisms. Similarly, bacterial cultures change in response to environmental stimuli that are relayed to intracellular signaling reactions that allow adaptation to and survival in various habitats. The signal transduction systems that determine cellular function and behavior are finely tuned circuitries composed of signaling proteins with equally sophisticated regulation. Using a multidisciplinary approach, we investigate these common, biomedically important, and intricate systems in two main research areas: bacterial biofilm formation and membrane trafficking in eukaryotes.
Most signaling proteins contain multiple domains and evolutionarily conserved modules. Previous studies, including those of my group, have revealed that protein domains do not work as separate entities but have evolved interdependently, developing unique features such as allostery and feedback mechanisms for regulation. The notion that protein domains are organized not as beads on a string but in a cooperative fashion is a primary foundation of our research, as coordinated functioning within complex regulatory proteins underlies some of the most fundamental physiological processes, whereas improper regulation accounts for a variety of diseases and pathologies. Our understanding of multi-domain proteins is still in its infancy, and detailed analyses of more of these proteins are needed to elucidate the key architectural and mechanistic principles of signaling reactions and networks.
We are examining these concepts in the bacterial signal transduction mechanisms that control biofilm formation, a process that contributes to infectious disease and pathogenesis. Central to the biofilm pathway is a bacterially unique second messenger, c-di-GMP, and enzymes for its biosynthesis and degradation (Figure 1). Although c-di-GMP was discovered in the late 1980s, its widespread impact on bacterial lifestyle and signaling has only recently been appreciated. My group was among the first to study systematically the structures and regulation of c-di-GMP signaling networks, revealing both conserved and protein-specific molecular mechanisms. These studies require a multidisciplinary approach combining structural data with the enzymatic parameters and cellular functions of these proteins. Highlights of our recent work include discoveries such as a novel c-di-GMP-dependent transcription factor that inversely regulates bacterial matrix production and motility and a conserved transmembrane signaling system that senses intracellular c-di-GMP levels to control extracellular processes. We expect our current and future studies to provide blueprints for interrupting biofilm formation, a predominant obstacle in the treatment of bacterial infections.
We are also investigating the molecular mechanisms and regulation of membrane fission and fusion in eukaryotes. In particular, we study peripheral membrane proteins that belong to the BAR domain superfamily and transmembrane proteins of the dynamin superfamily of GTPases that are central to the formation of the tubular endoplasmic reticulum (ER) network. We are elucidating the mechanisms driving topology changes within biological membranes that underlie many of the most fundamental cellular events. Aberrant membrane structure and dynamics in these processes can have severe biological and biomedical consequences, particularly in neurological and neurodegenerative diseases.
Common approaches in both projects include the structural characterization of proteins using X-ray crystallography and small-angle X-ray scattering (SAXS), in addition to several other biochemical and biophysical approaches. We use structural biology as a tool to generate new hypotheses. Functional in vitro and cellular assays complement these studies and validate conclusions drawn from molecular models.