You are here:Bert Van den Berg's Research Interests

Bert Van den Berg's Research Interests

I am Professor of Membrane Protein Structural Biology and joined the Institute of Cellular and Molecular Biosciences at Newcastle University in January 2013. Prior to coming to the UK, I was a faculty member within the Program in Molecular Medicine at UMass Medical School in Worcester, Massachusetts. The main focus of my research is to understand how small molecules are transported across the outer membrane (OM) of Gram-negative bacteria. For this, we determine the three-dimensional structures of bacterial integral OM proteins by X-ray crystallography. Those structures are then used, in combination with functional data obtained from biochemical experiments, to propose models that can be tested experimentally. Since becoming a group leader in 2004, my laboratory has solved the structures of a number of OM proteins. Please have a look at our colorful structure gallery for a selection of solved structures (Fig. 1). The gallery illustrates nicely that the beta-barrel scaffold of OM proteins is extremely versatile and supports many different folds and functionalities. In addition to OM uptake channels, we have solved structures of an OM protease (Pla), a full-length autotransporter (EstA) and the OM component of a metal efflux pump (CusC).

Figure 1 Cartoon representations of solved OM protein structures, using rainbow coloring with the N-terminus blue.

The location of the OM is shown by the grey bars. The transport substrates are listed in parentheses. Top row (left to right): E. coli Tsx (nucleosides), E. coli FadL (long chain fatty acids), P. putida TodX (toluene), P. aeruginosa FadL (paraffins) and E. coli OmpW (unknown, possibly hydrophobic molecules). Middle row: P. aeruginosa OccD1 (basic amino acids), Y. pestis Pla, P. aeruginosa EstA, A. baumannii CarO (basic amino acids) and E. coli CusC. Bottom row: P. aeruginosa OprO (polyphosphates), P. putida OprB (monosaccharides) and P. putida TcpY (chlorophenols). With the exception of OprO and CusC, all structures were solved using SAD or MIR approaches.


At the moment, our research on OM proteins is focused around two major themes:

1. Transport of hydrophobic molecules.

Uniquely, the OM of Gram-negative bacteria forms a very efficient barrier for the permeation of hydrophobic molecules, due to the presence of the lipopolysaccharide "sugar coat" layer on the outside of the cell. As a consequence, the uptake of hydrophobic molecules such as long-chain fatty acids (LCFA) and aromatic hydrocarbons requires special uptake proteins. The best-known examples are FadL channels, which are found in most Gram-negative bacteria. We have determined crystal structures of several FadL channels, including a number of site-directed mutant proteins. The structures show that the lumen of the FadL barrel is occluded by a globular plug domain, which is formed by the N-terminal ~40 residues of the protein (Fig. 2). The structural data, together with in vivo transport assays, demonstrated that E. coli FadL transports its LCFA substrates according to a novel, "lateral diffusion" mechanism. The decisive step in transport is the lateral movement of the substrate, through an opening in the wall of the channel, into the outer membrane (Fig. 2). Moreover, transport requires ligand-induced conformational changes in the plug domain, suggesting that FadL is a transporter rather than a channel.

Future research on FadL-mediated transport (supported by the National Institutes of Health) will focus on the uptake of environmental pollutants ("xenobiotics") during biodegradation. Most pollutants (e.g. aromatic hydrocarbons) are hydrophobic and require dedicated transport channels for uptake and subsequent metabolization in the cytoplasm. Our model system is Pseudomonas putida F1 (PpF1), a versatile, well-characterized biodegrader capable of metabolizing mono-aromatic hydrocarbons (MAH) such as benzene and toluene. PpF1 has three FadL orthologs, two of which have been crystallized by my lab (TodX and CymD). Interestingly, the channels that transport toluene (TodX, CymD) do not transport LCFA (Fig. 2), and vice versa. Explaining this difference in substrate specificity is one of the goals of our research. In addition, we would like to answer the following questions:

1. What is the transport mechanism for MAH? Preliminary data suggest that the mechanism might be different from lateral diffusion. 2. How important are FadL channels under conditions that resemble the natural environment? To answer this question we will collaborate with the chemical engineering group of Roseanne Ford at the University of Virginia. 3. Which cellular adaptations are required for growth of PpF1 on MAH? Given the fact that MAH make membranes leaky, we are especially interested in the identification of changes related to (phospho)lipid metabolism.

Figure 2 Summary of our work on FadL channels.

Experimental techniques that are used in the lab include recombinant DNA technology, membrane protein expression and purification, in vivo and in vitro substrate transport and binding assays, and protein structure determination by X-ray crystallography. In addition we will utilize microcosms and genome-wide random mutagenesis combined with deep sequencing to answer questions 2 and 3 listed above.

Because FadL orthologs are found in many biodegrading bacteria, our research is relevant for the bioremediation of xenobiotics within the environment.

    Lepore B.W., Indic M., Pham H., Hearn E.M., Patel D.R., van den Berg B. (2011) Ligand-gated diffusion across the bacterial outer membrane Proc. Natl. Acad. Sci U S A. 108(25):10121-6[Pubmed]
    van den Berg B. (2010) Going forward laterally: transmembrane passage of hydrophobic molecules through protein channel walls Chembiochem. 11(10):1339-43[Pubmed]
    Hearn E.M., Patel D.R., Lepore B.W., Indic M., van den Berg B. (2009) Transmembrane passage of hydrophobic compounds through a protein channel wall Nature 458(7236):367-70 [Pubmed]
    Hearn E.M., Patel D.R., van den Berg B. (2008) Outer-membrane transport of aromatic hydrocarbons as a first step in biodegradation Proc. Natl. Acad. Sci. U. S. A. 2008 Jun 24;105(25):8601-6 [Pubmed]

2. Understanding OM antibiotics uptake.

The increasing emergence of pathogenic (Gram-negative) bacteria that are resistant towards antibiotics represents a big future threat for public health, a situation that has recently been likened to a "ticking time bomb" and a possible "apocalypse" by the chief medical officer of England (http://www.bbc.co.uk/news/health-21702647). New antibiotics are therefore urgently needed. Despite this pressing need, very few new antibiotics have reached the market in the last decade, owing to the huge problems and risks in drug design.

Intracellular drug concentrations depend on the balance between influx, efflux and enzymatic degradation. Of these processes, influx and efflux of antibiotics are poorly understood. It is clear, however, that the successful design of effective antibacterials requires detailed insights in the basic biology of influx and efflux. In Gram-negative bacteria, the OM is the first (and frequently only) barrier encountered by antibiotics; consequently, drugs need to pass through OM channels in order to enter the cell. Indeed, changes in the levels of functional OM channels have been linked to resistance in many cases. We study the transport of antibiotics through OM diffusion channels to aid the design of drugs with efficient permeation properties. We are focusing on the channels of Pseudomonas aeruginosa (Pa) and Acinetobacter baumannii (Ab), two pathogens that are notorious for their resistance towards antibiotics. This is due to the extremely low permeability of the OM, which in turn is caused by the restrictive nature of the OM transport proteins. Pa contains ~30 OM uptake channels, whereas Ab may possess ~10-15.

The Occ (formerly OprD) family of substrate-specific OM channels is likely responsible for the uptake of the majority of water-soluble compounds in Pa and Ab. We have solved the structures of 14 out of the 19 Pa family members and have performed biochemical and biophysical characterizations for a number of them (in collaboration with Liviu Movileanu, Syracuse University) (Fig. 3). Together, the data show that Occ proteins transport substrates with a carboxyl group (hence the name Outer membrane carboxylate channels), with OccD subfamily members preferring linear compounds with a net positive charge (e.g. basic amino acids) and OccK family members preferring cyclic compounds with a net negative charge (e.g. benzoate) (Fig. 3). Insights into mechanisms of substrate transport have to come from co-crystal structures, and we have just published the first structure of an Occ channel-substrate complex (Fig. 3). We have also determined the first structure of an OM protein from Ab, CarO. Interestingly, the CarO structure shows a channel that is too narrow to explain the reported transport of amino acids and carbapenem antibiotics by this protein (Fig. 3).

Figure 3 Overview of our work on Pa and Ab OM channels.

Our future work on this project will take place within an exciting, recently established EU consortium consisting of a number of academic labs, small biotech firms and big pharma (http://www.imi.europa.eu/content/translocation). A major focus of my group will lie on characterization (co-crystal structures, binding/transport profiles) of the interactions of antibiotics with channels that are important and/or highly expressed during infection. Those channels will be identified using OM proteomics (Dirk Bumann, Biozentrum Basel). Other close collaborators within the consortium include electrophysiologists (Mathias Winterhalter, Jacobs-University Bremen) and computational biologists (Ulrich Kleinekathoefer, Jacobs-University Bremen and Matteo Ceccarelli, University of Cagliari). The overall goal of the project is to obtain atomistic, quantitative descriptions of antibiotics passage through relevant OM channels, exemplified by a movie from a simulation of ampicillin translocation through E. coli OmpF. The obtained insights will likely aid the design of novel antibiotics with superior permeation properties.

Movie legend

Simulation of ampicillin translocation through E. coli OmpF, viewed from within the plane of the membrane. Note the orientation of the antibiotic as it passes through the constriction of the channel, with electronegative groups (red) on the antibiotic interacting with positively charged side chains (blue) in the porin channel (movie provided by Matteo Ceccarelli, University of Cagliari).

    Biswas S., Mohammad M.M., Patel D.R., Movileanu L., van den Berg B. (2007) Structural insight into OprD substrate specificity Nat. Struct. Mol. Biol. 14(11):1108-9[Pubmed]
    Biswas S., Mohammad M.M., Movileanu L., van den Berg B. (2008) Crystal structure of the outer membrane protein OpdK from Pseudomonas aeruginosa Structure 16(7):1027-35[Pubmed]
    Eren E., Vijayaraghavan J., Liu J., Cheneke B.R., Touw D.S., Lepore B.W., Indic M., Movileanu L., van den Berg B. (2012) Substrate specificity within a family of outer membrane carboxylate channels PLoS Bio[Pubmed]
    Eren E., Parkin J., Adelanwa A., Cheneke B., Movileanu L., Khalid S., van den Berg B. (2013) Towards understanding the outer membrane uptake of small molecules by Pseudomonas aeruginosa. J. Biol. Chem. [Epub ahead of print][Pubmed]

Besides OM proteins, the lab also has an interest in helical bundle membrane proteins, in particular those involved in quorum sensing signal transport and integral membrane desaturases/hydroxylases. To this latter end we are also expressing membrane proteins in the yeast Saccharomyces cerevisiae.

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Highlights

February 2014
JBC Best paper of the year 2013 awarded to Pr. Rick Lewis
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January 2014
2014 is the year of Crystallography
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January 2014
CCP4 Study Weekend
3-5th of January, Nottingham University Read more...

Contact Info

Newcastle Structural Biology Laboratory
ICAMB, Medical School University of Newcastle Framlington place NE2 4HH, Newcastle upon Tyne, UK

Phone: (191) 222-7436
Fax: (191) 222-7424