In-depth structural characterization of the tetraspanin CD81

Every human cell is encased by a cell membrane that separates the cell contents from its surroundings. Proteins embedded in this membrane act as gates to allow molecules to enter and exit cells; they also mediate the interactions that occur between a cell and its environment. This means that membrane proteins are involved in many of the most fundamental processes in normal cell function; when these processes fail, diseases result. It is no surprise, then, that the top ten best-selling small molecule drugs of all time all target membrane proteins.

There are many different membrane proteins in any given cell, grouped into over 1,500 families, each with many members. In order to study any of them in detail, it is important to understand their three-dimensional structures. Central to this is a technique called X-ray crystallography that allows scientists to obtain a detailed view of how the atoms within a protein are arranged, providing a framework for further study. Scientists use this framework to investigate how the protein functions, bringing new levels of understanding to how cells work in health and disease, and providing knowledge to develop new drugs.

Tetraspanins are membrane proteins that function by interacting with a wide range of other membrane and soluble proteins, thereby affecting how cells signal, interact, change shape and move. Remarkably, tetraspanins are also involved in the process of infection for a wide range of diseases. However, because there is no known structure of any full-length tetraspanin family member, the mode of action of tetraspanins in these essential processes is not understood, leaving a major gap in our knowledge of cell biology.

Obtaining the structure of any membrane protein is a major scientific challenge: It is necessary to remove the protein from the cell membrane which often results in the protein becoming so unstable that it cannot be used to make the crystals required to perform X-ray crystallography. Consequently, we know very little about many membrane protein families with important biological functions. We have now overcome this crystallization challenge for the tetraspanin, CD81.

Human CD81 is one of the best understood tetraspanin family members and is the subject of our proposed research. It has well-established roles in how cells interact with each other, the immune response and fertilization. Notably CD81 is a receptor for some very important human pathogens including influenza, human immunodeficiency virus, the malarial parasite, T-cell lymphotropic virus type 1 and hepatitis C virus (HCV). It may also be a tumour promoter. Central to CD81 function (and to that of all tetraspanins) is its ability to form extensive interactions with itself and other proteins; however, we don't know what these structures look like and therefore lack the framework for further study, mentioned above.

The first aim of the research outlined in our proposal is to solve the three-dimensional structure of CD81. We have made excellent progress towards this goal, having crystallized CD81 and collected X-ray diffraction data.

We have also teamed up with scientists in France who can make soluble forms of the HCV protein, E2, that binds CD81. The second aim of our project is to make an HCV-E2/CD81 complex so we can characterize it and solve its structure; this will allow us to learn more about how CD81 interacts with other proteins. We believe we are the only team in the world that has all the tools to take on this challenge.

Brand new developments in structural biology (e.g. high-resolution electron microscopy) have enabled us to devise a third aim, which is to look at these structures in the cell membrane (by electron tomography), linking our atomic level structural data to what is actually happening in the cell.

Studying the structure of CD81 at this level of detail will allow us to begin to understand how tetraspanins work in health and disease.

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.