Multi-Pole Approach to Structural Science

May 10 - 13, 2015

Crossing membranes: beta-cyclodextrin-gemini surfactant and the type II secretion system
Pawel Grochulski, Masoomeh Poorgohorban, Ildiko Badea, Elizabeth Vanderlinde, Darek Martynowski , Peter Howard and Lee Wilson

Biological membranes can be crossed by ions, small molecules and proteins. There are two types of transport through biological membranes, passive and active transport. In the first type the transport is driven by diffusion, whereas in the latter case it is facilitated by other mechanisms. For example, the transport is by endocytosis in the case of the gemini-based drug delivery system and via a sophisticated protein machinery in the case of the type II secretion system (T2SS).

When designing a drug delivery system, one option would be to transport genetic material or an insoluble drug to a specific site inside the cell through a membrane using nanoparticles. In this process, the cell membrane engulfs the nanoparticle, forms a complete sphere around it, and then draws the membrane-bound vesicle, called an endosome, into the cell. Drugs loaded into nanoparticles can thereby be internalized efficiently by a large variety of cells, including cancer cells. Development and evaluation of novel β-cyclodextrin-gemini based nano-delivery systems for topical treatment of melanoma using synchrotron techniques will be presented. This includes the application of synchrotron WAXS (chemical crystallography, powder diffraction), SAXS and NMR in the characterisation of the system.

From a different perspective, transport of protein toxins and enzymes out of Gram-negative bacterial cells is performed via the type II secretion system, composed of 12-16 proteins depending on the species. Vibrio vulnificus utilizes the T2SS to translocate extracellular proteins from the periplasmic space across the outer membrane through a megadalton complex called the secretin. General secretion pathway (Gsp) proteins GspA and GspB are required for outer membrane localization and assembly of GspD, the protein that forms the secretin. The GspAB complex interacts with both peptidoglycan (PG) and the secretin to allow transport of proteins out of the cell. In V. vulnificus GspA and GspB are fused into one protein, EpsAB, whose crystal structure will be discussed. Another component of the T2SS in Aeromonas hydrophila is the inner membrane ExeC protein. The periplasmic domain of ExeC in A. hydrophila is composed of an N-terminal HR domain and a C-terminal PDZ domain. We have solved the crystal structure of the PDZ-domain expressed with a 6-His-tag at the C-terminus. Interactions of the 6-His-tag within the crystal structure of the PDZ domain may give some indication as to how the PDZ domain interacts with other components of the T2SS machinery. We also demonstrated that in A. hydrophila the PG-AB complex facilitates assembly of the secretin through direct interaction between ExeB and ExeD.