Abstract
FVIII is a multi-domain protein organized in a heavy and a light chain, and a B-domain whose biological function is still a matter of debate. The 3D structure of a B-domain-deleted FVIII variant has been determined by X-ray crystallography, leaving unexplained the functional nature of the flexible B-domain which could play an important role in the structure-function relationship since it is removed during the activation process. To obtain clues on the function of the B-domain, the morphology of full-length FVIII and its isolated domains was determined in the absence or presence of Ca2+. Recombinant full-length FVIII, the purified heavy chain, light chain and B-domain as well as B-domain-deleted rFVIII were analysed in buffers of different Ca2+ concentrations by atomic force microscopy. In the absence of Ca2+, FVIII appeared as a globular molecule, whereas at high amounts of Ca2+ up to 50-nm long tail structures emerged. These tails could be identified as unravelled B-domains, as images of isolated B-domains showed the same morphology and heavy chains which include the B-domain were also rich of tails, whereas the isolated light chains and B-domain-deleted FVIII lacked any deviation from a globular shape. The images further suggested that the B-domain interacts with the light chain particularly at low Ca2+ concentrations. Our results show a Ca2+-regulated conformational change of the B-domain in the context of full-length rFVIII. As the B-domain tightly associated with the core of the FVIII molecule under low Ca2+-concentrations, a stabilizing function on FVIII under non-activating conditions may be proposed.
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Conflict of interest
H. Rottensteiner, G. Schrenk, C. Fiedler, F. Scheiflinger and P. L. Turecek are full time employees at Baxter Innovations. K. Bonazza, G. Allmaier and G. Friedbacher declare that they have no conflict of interest.
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Bonazza, K., Rottensteiner, H., Schrenk, G. et al. Ca2+ concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy. Anal Bioanal Chem 407, 6051–6056 (2015). https://doi.org/10.1007/s00216-015-8778-z
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DOI: https://doi.org/10.1007/s00216-015-8778-z