title: Ca(2+) concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy.
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 Ca(2+). 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 Ca(2+) concentrations by atomic force microscopy. In the absence of Ca(2+), FVIII appeared as a globular molecule, whereas at high amounts of Ca(2+) 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 Ca(2+) concentrations. Our results show a Ca(2+)-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 Ca(2+)-concentrations, a stabilizing function on FVIII under non-activating conditions may be proposed.
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-IAC, 1060, Vienna, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Bonazza
authors.forename: Hanspeter
authors.initials: H
authors.lastname: Rottensteiner
authors.forename: Gerald
authors.initials: G
authors.lastname: Schrenk
authors.forename: Christian
authors.initials: C
authors.lastname: Fiedler
authors.forename: Friedrich
authors.initials: F
authors.lastname: Scheiflinger
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.forename: Peter L
authors.initials: PL
authors.lastname: Turecek
authors.forename: Gernot
authors.initials: G
authors.lastname: Friedbacher
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D018625:Microscopy, Atomic Force; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002413:Cations, Divalent; D011994:Recombinant Proteins; D005169:Factor VIII; D002118:Calcium