Molecular basis and selectivity of signaling and transport across lipid membranes

Membrane proteins represent over half of drug targets due to their accessibility and diverse roles in mediating interactions with and responses to the environment. My research program focuses on uncovering the molecular mechanisms and selectivity determinants of membrane proteins that mediate critical cellular processes such as transport, signaling, and specialized metabolic pathway regulation. To achieve this, we combine experimental techniques like X-ray crystallography and single-particle cryogenic electron microscopy with functional assays, both in vitro and in cells. We also use computational approaches, such as all-atom molecular dynamics simulations to investigate protein dynamics and bioinformatic analyses to investigate the evolution of protein families. Recently, we have expanded from structure-guided protein engineering to large-scale mutational screens, providing comprehensive datasets that enhance our understanding of structure-function relationships. Over the next five years our research will focus on the following three bacterial membrane protein systems—all of which contain common transporter folds that have evolved to perform diverse specialized functions: Nramp Transporters: Nramps, part of the LeuT-fold transporter superfamily, are metal ion transporters found across all kingdoms of life. In the past 10 years, we developed a detailed understanding of Nramp function using a bacterial homolog. Leveraging this rigorous foundation, we will use large-scale mutational screens to identify determinants of metal ion selectivity. These findings will clarify the evolution of substrate selectivity among Nramps and provide insight into strategies organisms use to avoid toxic metal accumulation. Type II Prodrug-Activating Peptidases: These bacterial peptidases, which include a type IV ABC transporter fold, selectively export and activate specialized metabolite toxins. Our recent elucidation of the structure and selectivity determinants of the type I colibactin-activating peptidase ClbP and expertise on ABC transporters prime our research on how the export and enzymatic activities of ZmaM are coordinated to activate zwittermicin. These insights could inform synthetic biology applications, including engineered microbial systems. TM-LuxR Regulators: Combining a Major Facilitator Superfamily (MFS) transmembrane domain with a LuxR- like DNA-binding domain, these transcriptional regulators are pivotal for extracellular sensing and transcriptional responses in gut microbiota. We aim to elucidate the structural basis for ligand specificity and regulatory mechanisms, shedding light on their roles in bacterial adaptation and host-microbe interactions. Biomedical Relevance: This research will provide foundational knowledge on important membrane protein families and how their members gain specializations. The insights gained on Nramp transporters could aid in mitigating metal toxicity and improving micronutrient homeostasis. Understanding prodrug-activating peptidases and TM-LuxR regulators will offer pathways for manipulating bacterial metabolism with potential implications for microbiome health and disease management. Project Number: 1R35GM161633-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: RACHELLE GAUDET | Institution: HARVARD UNIVERSITY, CAMBRIDGE, MA | Award Amount: $508,111 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award B Study Section[MRAB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260312