Extensive conformational sampling in a ternary electron transfer complex

Abstract

Here we report the crystal structures of a ternary electron transfer complex showing extensive motion at the protein interface. This physiological complex comprises the iron-sulfur flavoprotein trimethylamine dehydrogenase and electron transferring flavoprotein (ETF) from Methylophilus methylotrophus. In addition, we report the crystal structure of free ETF. In the complex, electron density for the FAD domain of ETF is absent, indicating high mobility. Positions for the FAD domain are revealed by molecular dynamics simulation, consistent with crystal structures and kinetic data. A dual interaction of ETF with trimethylamine dehydrogenase provides for dynamical motion at the protein interface: one site acts as an anchor, thereby allowing the other site to sample a large range of interactions, some compatible with rapid electron transfer. This study establishes the role of conformational sampling in multi-domain redox systems, providing insight into electron transfer between ETFs and structurally distinct redox partners.

Extensive Domain Motion and Electron Transfer in the Human Electron Transferring Flavoprotein·Medium Chain Acyl-CoA Dehydrogenase Complex

“…The ETF docking site is relatively simple, and because of the conformational flexibility of domain II, the relative positioning of the docking site and the electron transfer site has few restraints. Removal of Leu␤ 194/195 either by mutagenesis or limited proteolysis (8) leads to impaired electron transfer, supporting further the hypothesis that all ETFs interact with their partners via the recognition loop. On the other hand, mutagenesis of the conserved Arg␣ 237/249 , or of residues proposed to transiently interact with Arg␣ 237/249 , has led to very varied effects on reaction rates with different partners.…”

“…This is clearly indicative of a "ball-and-socket" type of motion, where MCAD and the ordered regions of ETF form the socket in which the FAD domain can apparently move freely within the conformationally allowed space. Similar molecular motions occur in the bacterial ETF⅐TMADH complex (8) and also within a single protein chain (e.g. the Escherichia coli 5Ј-nucleotidase (22)).…”

“…An MCAD dimer should therefore be regarded as the partner of ETF; the N-terminal domain of one monomer binds the recognition loop, whereas the C-terminal FAD-binding domain of the other monomer hosts the moving FAD domain. Molecular dynamics calculations were used to systematically sample the position occupied by the FAD domain in the bacterial ETF⅐TMADH complex (8). These calculations suggest that the FAD domain occupies a large number of different positions, some of which show increased electronic coupling between the isoalloxazine ring of the ETF cofactor and the 4Fe-4S center of TMADH by reducing the distance to Ͻ 14 Å.…”

“…Recent models constructed using the known crystal structures of human ETF and its partner human MCAD have suggested that electron transfer from MCAD to ETF requires rotational movement of the ETF FAD domain (7). Also, the recent structure determination of ETF from the bacterium Methylophilus methylotrophus in complex with its partner trimethylamine dehydrogenase (TMADH) reveals a highly dynamic proteinprotein interface in which the FAD domain of ETF samples the available space within the complex (8). This diffusional motion within the complex is restricted by the static component of the interaction: a small hydrophobic interface centered on the ETF conserved residue Leu␤ 194 .…”

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

“…2b). This model is consistent with the proposed role of the same region in the structurally related cytochrome P450-BM3 (Sevrioukova et al, 1999) and reminiscent of those found in multiple redox centers containing proteins (Zhang et al, 1998,Lennon et al, 2000,Leys et al, 2003. Additionally, this swinging FMN domain mechanism would account for the slow rate of inter-module electron transfer in NOS.…”