Research Synopsis
Proteins and small molecules are translocated across lipid bilayers by integral membrane proteins that span the bilayer and facilitate translocation. My lab centers around determining the atomic resolution structure of membrane proteins and complexes that function to transport materials across lipid bilayers. The primary focus of research in the lab will be on ion transport, with an initial emphasis on phosphate and iron uptake mechanisms within pathogenic bacterial species including the Pseudomonadaceae and Neisseriaceae families. In addition to the structural and biochemical characterization of these ion transport system components, research in my lab will examine the membrane protein complexes that facilitate the proper insertion and assembly of these membrane protein transport components.
Biochemical and structural techniques used in this research
TopPhosphate specific transport and the Pho Regulon within Pseudomonas aeruginosa
Phosphate plays an essential role in nearly all metabolic, catabolic, and signaling events within virtually all living organisms. As such, the trafficking of phosphate anions is an immensely important function, yet the specific mechanism utilized to transport inorganic phosphate molecules across a lipid bilayer remains unclear. The Gram negative bacterial cell utilizes a double membrane system and employs a plethora of channels that can be constitutive or specifically expressed in its outer membrane (OM) in response to various environmental conditions. Most bacteria, including Pseudomonas react to environmental conditions of low phosphate by turning on a gene regulon leading to the expression of a number of phosphate trafficking proteins (Figure 1). This regulation is mediated by regulatory proteins anchored in the inner membrane (PhoR), that sense phosphate levels through an as yet uncharacterized interaction with the inner membrane transport complex.
Figure 1: Pho Regulon Pathway
Amongst these up-regulated proteins are structural proteins designed to scavenge phosphate from the surrounding environment including: OprP (a phosphate specific outer membrane transporter), PhoS (a phosphate specific periplasmic binding protein) and PhoABC (a phosphate specific inner membrane ABC importer). We have begun to structurally characterize all of the components of this transport pathway and delineate the mechanism of transport in molecular detail through a combination of crystallography, site-directed mutagenesis, biochemical, and functional characterization.
TopIron acquisition through the bacterial transferrin receptor
In the vertebrate host, the level of free extracellular iron is well below that required to support the growth of bacterial pathogens, largely owing to the iron-sequestering effects of iron-binding glycoproteins transferrin and lactoferrin. Successful bacterial pathogens have developed high-affinity iron uptake systems capable of acquiring iron from transferrin and lactoferrin. Members of the Neisseriacea and Pasteurellaceae family including Neisseria meningitidis and Haemophilus influenza possess receptors consisting of the surface exposed lipoprotein TbpB and the integral outer membrane protein TbpA that bind transferrin and are involved in the retrieval and transport of iron across the outer membrane. Within the periplasm, the ferric binding protein, FbpA, binds iron and escorts it to the inner membrane ABC transporter where it is transported into the cytoplasm (Figure 2).
Figure 2: Iron Acquisition via the Bacterial Transferrin Receptor
The structural information gained about the conserved interactions between these essential outer membrane proteins and transferrin will represent the target regions for the development of broad-spectrum vaccines against these highly infectious bacterial pathogens.
TopThe SLC26 Anion Transporter, YchM
The human solute carrier SLC26 family of anion transporters consists of 11 members that are expressed in polarized cells in organs such as the kidney, pancreas, intestine and liver where they mediate sulfate, chloride and bicarbonate transport across the plasma membrane. The SLC26 transporter family is widely-expressed and includes SulP transporters that are commonly found in bacteria, fungi and plants where they mediate sulfate or bicarbonate transport. In collaboration with the Reithmeier Lab we will investigate the structural and biochemical activities of the YchM transporter from E. coli, which is homologous to SLC26 family of bicarbonate transporters within humans. Recently we determined the high-resolution crystal structure of the STAS domain from E. coli YchM isolated in complex with acyl-carrier protein (ACP), an essential component of the fatty acid biosynthesis (FAB) pathway (Figure 3).
Figure 3: The STAS domain of YchM functions in Fatty Acid Metabolism
We concluded that YchM, a bacterial member of the SLC26 family of anion transporters, is linked to fatty acid metabolism via a direct interaction with ACP. The major goal of our work will be to define structure of the membrane protein YchM in order to illuminate the mechanism of bicarbonate transport and its role in fatty acid metabolism.
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