Abstract
Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.
Highlights
Human electron-transferring flavoprotein (ETF)1 is a ubiquitous electron carrier that interacts with at least 10 different dehydrogenases, some of which are structurally distinct [1]
We show that mutation of the conserved Glu-165 in human electrontransferring flavoprotein (ETF) leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase
And in sharp contrast to the observed disorder in wild-type ETF complexes, the crystal structure of the E165A ETF1⁄7MCAD complex reveals the FAD domain restricted to a single, productive conformation
Summary
Human electron-transferring flavoprotein (ETF) is a ubiquitous electron carrier that interacts with at least 10 different dehydrogenases, some of which are structurally distinct [1]. Notwithstanding obvious differences in the structure of trimethylamine dehydrogenase and MCAD, the structure of the protein-protein interfaces of the trimethylamine dehydrogenase1⁄72ETF and MCAD1⁄7ETF complexes are remarkably similar Both ETF partners present a shallow concave surface with the redox cofactor buried beneath. A residue located near the center of this surface is proposed to interact transiently with the conserved residue Arg-249␣ (human numbering) found in the ETF FAD domain This stabilizes productive conformations for electron transfer from the primary dehydrogenase to ETF. The combination of a simple and relatively robust mode of interaction centered on the leucine anchor, and of the ensuing conformational sampling of the FAD domain, explains how ETF can interact with several structurally distinct partner proteins. We propose Glu-165 plays an essential role in ensuring high complex dissociation rates by stabilizing distinct and non-productive conformations of the FAD domain and postulate conformational sampling involves random interaction of the FAD domain residue Arg-249␣ with key residues on the proteinprotein interface
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