Molecular basis and selectivity of signaling and transport across biological membranes

Cells interact with their environment via their membrane proteins, and these interactions shape the rapid evolution of these proteins. Cell surface localization also makes membrane proteins accessible drug targets; well over half of approved drugs target membrane proteins. Membrane proteins function as receptors, transporters, and enzymes, and their molecular mechanisms entail coordinated conformational changes that are triggered by specific interactions with ligands and substrates. Several membrane protein folds are ubiquitous in genomes spanning the tree of life, suggesting that they arose early in evolution and are highly adaptable. We are generally interested in investigating the connections between structure, function, and evolution of membrane proteins.

Approaches

We use a variety of cell-based and in vitro biochemical assays, mutational scanning, cryogenic electron microscopy, X-ray crystallography, and computational techniques (e.g., molecular dynamics simulations and bioinformatics analyses) to discover how these important proteins function in cells. Below are brief descriptions that illustrate our approaches and goals for specific protein systems.

Systems

Nramp transporters

Metals like iron and manganese are essential to physiological processes such as oxygen transport and energy metabolism. Nramps (Natural Resistance-Associated Macrophage Proteins) are transition metal transporters found in all kingdoms of life. They have a finely tuned selectivity for transition metals over similar divalent cations and a noncanonical mechanism for kinetically coupling metal and proton import. We are interested in these proteins for their key roles in physiology and disease and more broadly as models for substrate selectivity and proton cotransport in transporters . We seek to understand, at atomic resolution, the molecular mechanism of metal-ion transport by Nramps throughout their conformational cycle and to map their fitness landscapes with respect to different metals and under different regulatory conditions. We do so with x-ray crystallography, biochemical assays, molecular dynamics simulations, sequence analyses, and deep mutational scanning.

Insect Gustatory Receptors

We are interested in insect gustatory receptors as an example of a large and highly diversified family of sensory ion channels. Invertebrate Gustatory Receptors (GRs) are a large and evolutionarily diverse family of sensory receptors known to play important roles in invertebrate taste, smell, and thermotransduction. Given the importance of these sensory modalities in host-seeking behavior in important human disease vectors like mosquitoes, GR family members serve as potentially powerful targets for vector control agents. We are using structural, biochemical, and biophysical tools to investigate key family members. We are also using bioinformatics, leveraging the large amount of sequence information, to understand the structure, function, and evolution of this family of ion channels. We are also interested in applying deep mutational scanning to understand how ligand selectivity can be tuned.

Prodrug-Activating Peptidases

Both our microbiome and environmental microbes provide a rich source of bioactive small molecules useful as drugs or drug precursors, with penicillin as a classic example. Yet there remains a huge untapped potential to use and manipulate microbial biochemical activities to improve human health, which requires knowledge of the macromolecular structures and mechanisms of these biosynthetic pathways. We investigate how an understudied family of peptidases activates important bacterial bioactive compounds ranging from underexplored antibiotics to cancer-causing toxins. These results will help develop chemical tools to study our microbiome and bioengineering efforts to produce new bioactive compounds.

12TM-LuxR Transcriptional Regulators

Gut microbes use dietary and host-derived metabolites as energy sources. We investigate a recently discovered family of transmembrane LuxR transcriptional regulators called 12TM-LuxRs. 12TM-LuxRs are especially common in gut-dwelling Coriobacteriia like Eggerthella lenta, which has dozens. 12TM-LuxRs are exquisitely selective and regulate many metabolic pathways and influence bacterial interactions with the host and the gut environment. 12TM-LuxRs combine an N-terminal Major Facilitator Superfamily (MFS) transmembrane domain believed to serve as the sensor with a C-terminal LuxR-type DNA-binding domain (DBD). TM-LuxRs have thus adapted a transporter fold to serve as a receptor. We aim to shed light on microbial adaptations that are potential targets to control gut microbial communities by answering the following questions: How has the MFS fold been co-opted for signaling in 12TM-LuxRs? What are the molecular determinants for the 12TM-LuxRs’ exquisite ligand selectivity?