The nature of ligand-induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants

Ligand-induced conformational change in transferrins has been studied by site-directed mutagenesis of human serum half molecule (N-lobe), X-ray absorption fine structure (XAFS) spectroscopy and X-ray solution scattering. Use of recent advances in data analysis has been made for extracting model-independent molecular shapes from X-ray solution scattering data for the intact, the half molecule and its mutants. Clear evidence is provided that the transferrin molecule (intact as well as N-lobe), in its apo and hole forms, exists for the majority of the time in well-defined specific conformations representing the "fully opened" and "closed" states of the molecule, respectively. Evidence is also provided for the existence of an additional conformation, referred to here as the "intermediate" conformation for simplicity, which is trapped in the case of some of the mutants in the iron-bound form. We suggest that domain closure in the transferrin molecule is a two-step process, with the intermediate conformation representing the first stage of domain closure (similar to 20 degrees hinge-twist of domain II). Our data are not inconsistent with the ligand-free molecule sampling the closed states occasionally (less than or equal to 10%) but are not in support of a continuous conformational search between the fully opened and closed states in the absence of iron. (C) 1998 Academic Press Limited.