Structure and function of Heparin Binding Hemagglutinin from Mycobacterium tuberculosis

Tuberculosis (TB) is a leading cause of death worldwide due to a single bacterium, Mycobacterium tuberculosis (MTB). Key aspects of TB pathogenesis include the ability of MTB to disseminate from the site of primary infection (primarily the lungs) to potentially any organ (Russel et al, 2010 Science, 328:852-856) via the interaction of MTB with epithelial cells. This process requires a crucial virulence factor called heparin-binding haemagglutinin adhesin (HBHA, Pethe et al., 2001 Nature 412, 190-194), a protein that is localised at the surface of MTB. In addition to the relevant function in TB, HBHA is considered a crucial target for diagnostic and treatment of TB, which is particularly crucial in view of the growing multi-drug resistance of MTB.

Currently the structure of HBHA is unknown. The challenging nature of this study is associated with the complex nature of HBHA that has prevented so far the successful employment of methods able to characterise protein structures. This lack of understanding has in turn hampered the characterisation of the mechanisms by which HBHA promotes the extrapulmonary dissemination of TB.

The proposal aims at the ambitious target of resolving the structure of HBHA and the functional mechanism by which this protein promotes the adhesion to epithelial cells within the mechanisms of extrapulmunary dissemination of TB. This is a milestone in TB research that requires overcoming significant experimental challenges. Our research proposal comes at a most opportune time since we now have all the tools, materials and background research to achieve this major goal. We will combine our research programmes in biomolecular nuclear magnetic resonance (NMR) spectroscopy and microbiology to generate a multidisciplinary investigation to reveal the structure, dynamics and topology of HBHA at the surface of MTB membranes as well as the details of the mechanism of adhesion to epithelial cells.

The impact of our interdisciplinary study will be significant on the wider academic community studying the underlying molecular mechanisms of TB infection and wider disciplines such as protein science, biochemistry, NMR spectroscopy, microbiology, cellular and molecular biophysics. It is anticipated that the outcomes of this research may directly translate into knowledge leading to new therapeutic and diagnostic approaches for TB.

This project aims at an interdisciplinary study of the structural basis of the epithelial cell adhesion by Mycobacterium tuberculosis (MTB) by the heparin-binding haemagglutinin adhesin (HBHA), a homodimeric protein localised at the surface of MTB. The challenging aspect of this ambitious characterisation is associated with the structural complexity of HBHA, which is composed of a membrane-associated domain (residues 1-25), a coiled coil region (residues 26-110) and an intrinsically disordered region (residues 111-199) having the function of sensing and binding epithelial cells via its heparin binding domain (residues 161-199).

To characterise this key protein, we will carry out an integrated investigation of solution and solid-state NMR (ssNMR) that will provide high-resolution data to refine the structures of the three HBHA domains (phase I). The membrane associated region will be studied using solid-state NMR, whereas coiled coil and heparin-binding domains will be investigated using solution NMR experiments that are tailored to the characterisation of folded and disordered protein states, respectively. These sparse NMR data will be integrated in an advanced refinement protocol that integrates tools tailored to the characterisation of the structure and dynamics of protein states (phase II).

Finally, in phase III we will probe the specific interaction of HBHA with epithelial cells using advanced NMR methods to characterise in detail weak and transient interactions between disordered proteins and complex cellular surfaces (Nature Communications, 2016, 7:12563). This study will identify the relationship between structure, dynamics and functional mechanism of HBHA.

The outcomes of this research will impact both the academic and the industrial sectors and will range from basic to applied sciences. The project will provide a key knowledge toward the identification of new therapies to combat Tuberculosis (TB). Moreover, the multidisciplinary research that will be carried out will enhance dramatically our ability to study other fundamental processes that are relevant to both academia and industry by extending the applicability of molecular spectroscopy to highly complex processes.

INDUSTRIAL BENEFICIARIES: The project is likely to have an impact on the pharmaceutical industry by advancing the use of NMR spectroscopy to understand complex molecular processes. Direct beneficiaries include those pharmaceutical companies working at the definition of therapeutic approaches to combat TB. More broadly, the interdisciplinary approaches here proposed will enhance our ability to characterise the mechanisms of heterogeneous biological processes, with key impact on many bio-industrial areas.

MEDICAL CHALLENGES IN TUBERCULOSIS: Understanding the molecular bases of dissemination of TB from the primary site of infection is a key goal for TB research. By characterising the structure and the functional mechanism of HBHA, the project will enable for new routes of diagnostics and drug discovery toward the identification of effective therapeutic molecules against TB.

DISSEMINATION: We will make every effort to ensure that research is disseminated widely to the research community by the Open Access publication in high-impact journals, presentation at international research meetings and the development of new collaborations. Furthermore, protocols and algorithms developed for the analysis of complex NMR data will be made available through our websites at Imperial College and via the Collaborative Computing project for NMR (CCPN: http://www.ccpn.ac.uk/ccpn). We are highly committed to make our findings available to the wider public. Lectures explaining how NMR spectroscopy assists in the drug development, TB research and scientific discovery will be made school-oriented presentations and Schools Open Days. The press offices at Imperial College will assist in disseminating discoveries via the popular science press and other media formats as proven in our track record.

TRAINING: In the proposed research we will employ and develop state-of-the-art methods of NMR spectroscopy to contribute in the wide area of structural biology. This provides an excellent platform to enhance training of PDRAs, PhDs as well as undergraduate students at the interface of biology, spectroscopy, medicine and chemical engineering.