In-depth structural characterization of the tetraspanin CD81

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Tetraspanins are membrane proteins that function by forming diverse, incompletely-understood oligomeric complexes. Our knowledge of the family is limited by the fact that (i) there is no high-resolution structure of any full-length member and (ii) the oligomeric status of the functional tetraspanin unit in cells is not known.

We have a unique opportunity to solve a high-resolution structure of one of the best characterized human tetraspanins, CD81, which is involved in cell-cell adhesion, cell proliferation, the immune response and fertilization. It also has an established role in infection by influenza, human immunodeficiency virus, the malarial plasmodium parasite, human T-cell lymphotropic virus type 1 and hepatitis C virus (HCV); CD81 may also be a tumour promoter.

We have crystallized CD81 from a homodimeric preparation (the dimer is hypothesized to be the functional unit of CD81). Our X-ray crystallographic dataset will allow us to solve the first structure of any tetraspanin.

Binding of the HCV-E2 glycoprotein to CD81 has been characterized in cell culture; the crystal structure of a soluble HCV-E2 ectodomain in complex with two antibody fragments was reported recently. Our biochemical data on the molecular determinants of the HCV-E2/CD81 interaction together with our ability to produce soluble HCV-E2 ectodomains and full-length CD81 uniquely enables us to investigate CD81 complexes in vitro using size exclusion chromatography, dynamic light scattering, multi-angle light scattering, surface plasmon resonance and crystallization.

We have generated a unique panel of anti-CD81 antibodies (using our recombinant CD81) that can distinguish different CD81 oligomeric states; electron tomography in cells will enable us to use these antibodies to understand how CD81 homodimers exert their biological function in membranes. This will complement our in vitro studies and bridge the resolution gap between the cellular and structural biology of CD81

The BBSRC policy news website states that the bio-based economy "encompasses a wide range of activities that use bioscience-based research or processes to produce products, food, fuel or therapies. Across Europe the sector already represents a market worth over 1.5 trillion Euros and more than 22 million people are employed in the bioeconomy".

The outputs of this project will directly contribute to the bio-based economy through their potential for commercial application: CD81 has a well-established role in infection by various pathogens including influenza, human immunodeficiency virus, the malarial plasmodium parasite, human T-cell lymphotropic virus type 1 and hepatitis C virus; it has also been proposed to be a tumour promoter. CD81 is involved in essential physiological processes including cell-cell adhesion, cell proliferation, the immune response and fertilization. An understanding of how CD81 forms homomeric and heteromeric complexes will therefore impact on our knowledge of many different proteins with fundamental biological functions as well as those involved in cancers and infectious diseases.

The outcomes of the proposed research will be exploited according to the "Pathways to Impact" document that accompanies this proposal and is likely to benefit the following non-academic beneficiaries:

1. Scientists in pharmaceutical and biotechnology companies - this work seeks to define the first structure of the tetraspanin family. We will also establish the oligomeric status of the CD81 functional unit. This knowledge will enable new drug targets to be defined and, in the long term, facilitate the development of allosteric ligands that can enter drug development pipelines;

2. UK economic competitiveness - No complex of a viral attachment protein together with an integral membrane receptor has been characterized in atomic detail to date; the results from this proposal will lead to the first such structure. This will be possible because of the expertise developed in this project on forming, stabilizing and characterizing HCV-E2/CD81 complexes. Potential exists for molecules or vaccine candidates to be developed by UK companies based on the new structural and functional understanding of CD81 complexes that will result from this project. In the long term, this will allow companies to create new jobs;

3. Patients - in the longer term, access to new and improved therapies for important human infections will be of direct benefit for patients, thereby improving the quality of life across the lifespan;

4. The scientists of tomorrow & their families- The primary impact of this research will be enhanced structural and functional understanding of a membrane protein (the tetraspanin, CD81) of broad biological and medical importance. Starting with our first display in 2016 (11th-20th March), we will build an exhibit to engage the public. We will show how microbes are used as biotechnological tools to make medically-important proteins that can be developed as drug targets. In the long-term, the exhibit will be used as part of Aston's ongoing links with Thinktank (Birmingham's science museum) which is directly opposite the Aston campus;

5. The researcher co-investigator, Dr Michelle Clare, and the PGRA on this proposal - MC generated preliminary data for this proposal during her BBSRC iCASE-funded PhD project and has established the experimental systems that underpin it. MC's exceptional crystal data are highly likely to lead to a high-impact publication within the first 18 months of the project. Supported by the high-quality portfolio of work, training by the investigator team and publications arising from the proposed project, (i) MC will be in a strong position to transition to independence by securing a personal fellowship at the end of the project and (ii) the PGRA will develop his/her employability by gaining sought-after EM, crystallization and other high-level technical skills.