Structural Biology and Biophysics of Large Cellular Assemblies
Co-workers: Dr. Beatrice Li, Dr. Anastassia Kantsadi, Ms. Julia Busch, Ms. Jemma Day, Ms. Jodie Ford, Mr. Samuel Bunt
With over 110,000 structures determined by X-ray crystallography and NMR, the universe of protein shapes is thoroughly explored. Less so are the interactions formed by these proteins, yet protein function is increasingly attributed to formation of stable or transient complexes. My group focuses on protein interactions underpinning cellular systems of medical relevance, which we characterise through a combination of X-ray crystallography, NMR and biophysical methods. We are particularly interested in centrioles, cell organelles implicated in developmental abnormalities and cancer, as well as in the malaria parasite and its interactions with human red blood cells.
Centrioles, the major component of animal centrosomes, must duplicate once per cell cycle in order to maintain their number. Abnormalities in this process can lead to ciliopathies, male sterility and cancer. It is therefore important to understand how these cell organelles assemble, and how their assembly is regulated. Genetic analysis uncovered a number of proteins, including SAS-6, SAS-4/CPAP, SAS-5/STIL and Cep135, and microtubules as being important for this process. We now need to place these components in the context of centriolar assembly, and to understand their mechanistic function.
Recently, we and co-workers showed that SAS-6 is the first structural component in the centriole assembly process. SAS-6 forms a large, multimeric framework that determines centriolar symmetry and allows other components to attach. CPAP, one of these components, adopts a structure suitable for elongating centrioles, while SAS-5 likely acts as a nucleation point that triggers centriole assembly. Much work remains, however, to understand how these components link with each other and how these linkages change as centrioles mature.
Malaria is virtually absent in the developed world but it still affects the lives of about half the world's population, with an estimated 200 million infections and 600,000 deaths in 2013. Most malaria deaths are caused by a single parasite species, Plasmodium falciparum, and many can be attributed to obstruction of small blood vessels by red blood cell clumps. Our research focus is on understanding how parasite proteins modify the host red blood cell so that its shape, flexibility and adhesion characteristics change.
P. falciparum is exceptional among malaria parasites in the number of proteins (~500 or 10% of total) it exports to its host cell. Some of these proteins help to establish novel membrane structures in the host, others interact with the red blood cell cytoskeleton, and others establish large protrusions on the host cell surface, called knobs. Over the last few years we have started linking the parasite adhesion receptor, PfEMP1, with components of the parasite exportome that assist pathogenic adhesion. In the process we determined the first structure of a novel protein family, unique to Plasmodia, called PHIST. We now try to understand how PfEMP1 and PHIST proteins link to the host cytoskeleton, and in so doing alter its mechanical properties.
To address these questions we take full advantage of extensive local infrastructure, such as the Oxford Protein Production Facility, the Diamond Light Source and the Oxford BioNMR Facility. We also collaborate extensively with leading laboratories in the UK and abroad. Projects typically combine biophysics, computational methods and in vivo functional assays, and they often allow lab members to visit collaborating groups to learn new methods.
Recent news items
Selected recent publications
1. Rogala KB, Dynes NJ, Hatzopoulos GN, Yan J, Pong SK, Robinson CV, Deane CM, Gönczy P, Vakonakis I (2015) The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation. eLife 4, doi: 10.7554/eLife.07410.
2. Oberli A, Slater LM, Cutts E, Brand F, Mundwiler-Pachlatko E, Rusch S, Masik MFG, Erat MC, Beck HP, Vakonakis I (2014) A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface. FASEB J 28, 4420-4433.
3. Hatzopoulos GN, Erat MC, Cutts E, Rogala KB, Slater LM, Stansfeld PJ, Vakonakis I (2013) Structural analysis of the G-Box domain of the microcephaly protein CPAP suggests a role in centriole architecture. Structure 21, 2069-77.
4. Hilbert M, Erat MC, Hachet V, Guichard P, Blank ID, Flückiger I, Slater L, Lowe ED, Hatzopoulos GN, Steinmetz MO, Gönczy P, Vakonakis I (2013) The Caenorhabditis elegans centriolar protein SAS-6 can form a spiral that is consistent with imparting a 9-fold symmetry. Proc. Natl. Acad. Sci. U.S.A. 110, 11373-8.
5. Mayer C, Slater L, Erat MC, Konrat R, Vakonakis I (2012) Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope. J Biol Chem. 287, 7182-9.
6. Kitagawa D, Vakonakis I, Olieric N, Hilbert M, Keller D, Olieric V, Bortfeld M, Erat MC, Flückiger I, Gönczy P, Steinmetz MO (2011) Structural Basis of the 9-Fold Symmetry of Centrioles. Cell 144, 364-75.
Cartoon showing how the nine-fold symmetric cartwheel model determines the overall symmetry of centrioles, drawn as ensembles of microtubule blades. An electron micrograph of C. reinhardtii SAS-6 protein with clearly visible ring structures is shown in the background.
Schematic representation of a complex between two major exported proteins of the malaria parasite, PHIST (in white) and PfEMP1 (green), combining X-ray and NMR (red surfaces) data, and computational modelling.
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