The previously unknown structure of the inhibitory platelet receptor, G6b-B has now been solved by Peak Proteins in complex with a glycan ligand similar to that found on perlecan. This surprisingly revealed a “head to tail” dimer around the glycan ligand with implications for how G6b-B signals to the intracellular environment.
Platelets are highly reactive, small anucleated cells produced in the bone marrow, spleen and lungs by megakaryocytes. They circulate in the blood stream and when they encounter a damaged blood vessel are activated and form a plug to stop bleeding. Their activation is tightly regulated to avoid hyperactivity, which could occlude blood vessels and consequent cardiovascular pathologies.
G6b-B is a key inhibitory cell surface receptor that is highly expressed on megakaryocytes and platelets. The molecular interactions that enable this inhibitory effect have previously been unknown, but are now revealed in exquisite detail in this landmark study. The full publication can be found here.
The study was led by Professor Yotis Senis from the University of Birmingham and involved a collaboration of 13 different organisations. Peak Proteins’ role was to determine the crystal structure of the extracellular portion when complexed to a glycan ligand. This presented multiple challenges that needed to be overcome.
Firstly, the covalently attached oligosaccharide side chains needed to be greatly simplified in order to give the protein a chance of forming an orderly crystalline array. This was achieved by the engineering and production of 11 different mutant proteins and their detailed characterisation by mass spectrometry. Over half of the mutant proteins were also evaluated in crystallisation screens, sampling many thousands of different conditions using automated procedures.
One protein was progressed, but further improvements were required involving co-crystallisation with an antibody Fab fragment and substantial crystallisation optimisation. Finally this gave crystals that diffracted sufficiently well at the Diamond Light Source Synchrotron facility in Oxford.
The structure of the G6b-B-Fab complex was initially solved by molecular replacement using a publicly available Fab structure as the search model. The early electron density for the G6b-B itself however was difficult to interpret and required multiple rounds of careful model building and refinement to eventually reveal the final structure of the G6b-B dimer as shown below.
The crystal structure reveals how G6b-B dimerises around the glycan ligand. This is of functional importance as many receptor proteins only function when they are dimers. The structure also reveals in atomic detail how the antibody binds the G6b-B, the so-called epitope map. This information has been incorporated into a patent filing from the University of Birmingham.
Professor Senis commented, “Having established the critical role of G6b-B in regulating platelet homeostasis, identifying the ligand and the molecular basis of this interaction became of paramount importance. Having personally no prior experience of crystallography, Peak Proteins came highly recommended by leading academics in the field”.
“Peak Proteins quickly ascertained the challenge and got to work. They were highly approachable, experienced and professional, and an excellent rapport was quickly established, keeping my team and I up-to-date on the latest findings, obstacles and solutions. They doggedly persevered from start-to-finish, achieving our objective more efficiently and economically than would have been possible otherwise. It was a high risk, but high reward. The findings were invaluable scientifically and for future drug discovery. Money well spent!”, continued Professor Senis.
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