EPSAM

Structural studies of the molecular basis of host-pathogen interactions in malaria

Lead Supervisor:  Prof Trevor Forsyth

Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis.  Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. Expression systems have been developed that allow biophysical studies of the two proteins. To pursue this, the University of the Witwatersrand is planning a collaboration with the Institut Laue Langevin (ILL), the European Synchrotron Radiation Facility (ESRF) Grenoble in France, and the Australian National University (ANU) to initiate an in-depth programme of research that will elucidate the molecular basis of this important interaction. The approaches will use the technical platforms available in the PSB, notably the HTX platform, MX beamlines, Deuteration Laboratory and SAXS/SANS.

Programme of work

This project will focus on the characterisation of the interaction between the 4.1 R protein and EBA-181. The following experimental strategies with be applied. They will exploit the availability of the expression systems developed by the Coetzer group, as well as facilities within the PSB, including the X-ray beamlines, the Deuteration Laboratory, the High Throughput Crystallisation (HTX) platform, the SANS/SAXS platform, the 1-D and 2-D NMR platforms, negative-stain and cryo EM facilities, as well as the Biophysical Characterisation platform. The overall strategy will be to apply the available complementary techniques in a way that should allow a technique-independent consensus on the structure, interactions and functional relationships of these proteins in the context of the host-pathogen interaction. The wide range of techniques available means that should some techniques finally not be applicable, the student will nonetheless obtain a set of structural results still highly suitable for a successful thesis project.

(a) Crystallisation of 4.1 R and of EBA-181 separately. As a matter of course, both proteins will be expressed and sent to the HTX crystallisation robots.

(b) Crystallisation of 4.1 R and EBA-181 as a complex. The 4.1R/EBA-181 co-complex will also be produced and sent to the HTX robots.

For both (a) and (b), crystallographic data collection will be carried out on the ESRF MX beamlines. If crystals of sufficiently large size can be grown, neutron crystallographic work will be carried out on the LADI-III diffractometer at the ILL.

(c) SANS and SAXS studies of the 4.1R/EBA-181 in solution. The PSB SANS/SAXS platform will be used to record smallangle solution scattering data from instruments D22 at the ILL and ID14.3 at the ESRF. For the SANS part of the work, selectively labelled components will be produced and used in a way that has now been widely established (Callow et al, 2007; Vijaykrishnan et al, 2010).

(d) Neutron and X-ray reflection studies of 4.1 R in whole membrane and in the presence of EBA-181. Since this system is heavily related to a membrane-bound complex, reflection experiments will carried out using the new FIGARO reflectometer at the ILL; again these will be complemented by analogous X-ray experiments. Suitably labelled neutron samples will be used to study the location of 4.1 R in the membrane and the changes that occur upon binding of EBA-181. (NMR measurements will be carried out using the 900MHz system in the PSB. 2H, 13C, 15N labelling will all be used for this and the information gathered will be cross-correlated with that obtained from the SANS/SAXS solution studies.

(e) Electron microscopy will be used as needed for both routine characterisation (negative stain EM) and detailed 3D work (cryo EM).