Section: Research

Bert van den Berg, Ph.D.

Academic Role: Assistant Professor

Faculty Appointment(s) In:
   Program in Molecular Medicine

Other Affiliation(s):
   Interdisciplinary Graduate Program

Structure and function of membrane transport proteins

Biological membranes form the interface between cells and their environment. Transport of molecules across this barrier is essential for all cells (for metabolite supply, for example). As a consequence, many (human) diseases, with cystic fibrosis as the best known example, are caused by mutations in membrane transport proteins. As such they are also of enormous medical importance, indicated by the fact that more than 70% of pharmaceutical drugs target membrane proteins.

Unfortunately, our understanding of how these proteins carry out their tasks is still rather limited. This is mainly due to a lack of detailed structural information. To date only ~70 unique membrane protein structures have been deposited in the Protein Data Bank (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html ). This is only 0.5% of all known protein structures, despite the fact that membrane proteins are thought to comprise ~25-30% of all open reading frames. This discrepancy is mainly due to the difficulties in obtaining milligram amounts of highly pure membrane proteins, required for structural studies. However, the increase in the number of solved membrane protein structures in recent years demonstrates that structure determination of membrane proteins, although challenging, is definitely feasible.

We are interested in determining the 3D structures of membrane transport proteins by X-ray crystallography. The structures are then used, in combination with functional data obtained from biochemical experiments, to propose mechanistic models that can be tested experimentally.

The research in my laboratory is centered around two areas:

1. Transport of hydrophobics

Little is known about how hydrophobic substances, such as long-chain fatty acids (LCFAs) and aromatic hydrocarbons destined for biodegradation, are transported across membranes. In principle, such compounds could cross membranes spontaneously by diffusion, but many pro- and eukaryotic cells have evolved regulated and highly efficient transport processes.

We recently determined crystal structures of the long-chain fatty acid transporter FadL, a conserved protein that transports LCFAs across the outer membrane (OM) of gram-negative bacteria (1). FadL forms a 14-stranded β-barrel that is occluded by a central hatch domain that can undergo conformational changes to form a channel. These structures have led to a model for LCFA transport across the bacterial OM (Figure 1).

Future work will focus on:

1. Biochemical characterization and crystallization of FadL mutants to test and further define the LCFA transport model (in collaboration with Paul Black, Albany Medical College). In addition, we intend to crystallize FadL proteins of pathogenic bacteria (most notably Omp P1 from Haemophilus influenzae). We intend to use the structural information to design novel antibiotics that enter bacteria via the FadL channel and interfere with central metabolic processes (for example phospholipid and lipid A biosynthesis).
   
2. Crystallization of the OM toluene transporter TodX from Pseudomonas putida, which is homologous to FadL. Transport of xenobiotics across the OM is likely to be a decisive step for biodegradation in gram-negative bacteria. Questions we would like to answer include: what makes the channel specific for aromatic hydrocarbons? Using structural information, would it be possible to improve transport efficiency or change the substrate specificity?
   
3. Characterization and crystallization of OmpW, a conserved OM protein of unknown function that is homologous to alkane transporters of biodegrading gram-negative bacteria (in collaboration with Paul Black, Albany Medical College).

2. Protein translocation

Protein translocation channels play central roles in all cells, since they are involved in secretion of soluble proteins across membranes and/or integration of integral membrane proteins. Despite many biochemical studies, most of the mechanistic aspects of protein translocation and membrane insertion are still poorly understood. We are studying the following systems:

1. SecY complex. This hetero-trimeric protein complex is universally conserved in all kingdoms of life and forms a protein-conducting channel in the plasma membrane, allowing polypeptides to be transferred across or integrated into membranes. Recently, the X-ray structure of an archaebacterial SecY complex was solved in the closed state (2), enabling us to propose a model for how this fascinating machine works (Figure 2). Future work (in collaboration with Tom Rapoport, Harvard Medical School) will focus on crystallization of an open channel and of a channel with a bound signal sequence.
   
2. Omp85.  Members of the Omp85 family are essential for cell viability, and are present in the OM of all gram-negative bacteria and in the outer mitochondrial membrane of eukaryotes (ranging from plants to humans). Emerging evidence suggests that Omp85 mediates integration of OM proteins, but how it performs this task is completely unclear. We seek to answer this by solving the Omp85 X-ray structure. To do this we will use the strategy of cloning/overexpressing a number of Omp85 homologues in order to obtain well-diffracting crystals.
   
3. Two-partner secretion (TPS).  Many large virulence proteins of pathogenic gram-negative bacteria are secreted by the TPS pathway. As the name implies, only two proteins are required for this process: the secreted exoprotein (TpsA) and the β-barrel transport channel in the OM (TpsB). After crossing the inner membrane via the SecY complex, TpsA is likely translocated across the OM in an unfolded state. Many aspects of the transport process are unclear: how is periplasmic TpsA kept soluble, how does targeting to the OM channel occur, and how is TpsA transported through the TpsB channel, given that no external energy is required? To answer these questions we plan to crystallize a TpsB channel and to reconstitute the transport process in vitro with purified components.

Office: Biotech 2, Suite 115
Phone: 508-856-1201
E-mail: Lambertus.VandenBerg@umassmed.edu
Keywords: Microbial Pathogenesis, Structural Biology, Biochemistry

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