3CLR

Crystal structure of the R236A ETF mutant from M. methylotrophus

3CLR の概要

• エントリーDOI: 10.2210/pdb3clr/pdb
• 関連するPDBエントリー: 1O97 3CLS 3CLT 3CLU
• 分子名称: Electron transfer flavoprotein subunit beta, Electron transfer flavoprotein subunit alpha, ADENOSINE MONOPHOSPHATE, ... (6 entities in total)
• 機能のキーワード: etf, tmadh, electron transfer, flavoprotein, electron transport, fad, transport
• 由来する生物種: Methylophilus methylotrophus; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 63825.61

構造登録者

Katona, G.,Leys, D. (登録日: 2008-03-20, 公開日: 2008-04-08, 最終更新日: 2023-08-30)

主引用文献

Burgess, S.G.,Messiha, H.L.,Katona, G.,Rigby, S.E.,Leys, D.,Scrutton, N.S.
Probing the dynamic interface between trimethylamine dehydrogenase (TMADH) and electron transferring flavoprotein (ETF) in the TMADH-2ETF complex: role of the Arg-alpha237 (ETF) and Tyr-442 (TMADH) residue pair.
Biochemistry, 47:5168-5181, 2008

PubMed Abstract: We have used multiple solution state techniques and crystallographic analysis to investigate the importance of a putative transient interaction formed between Arg-alpha237 in electron transferring flavoprotein (ETF) and Tyr-442 in trimethylamine dehydrogenase (TMADH) in complex assembly, electron transfer, and structural imprinting of ETF by TMADH. We have isolated four mutant forms of ETF altered in the identity of the residue at position 237 (alphaR237A, alphaR237K, alphaR237C, and alphaR237E) and with each form studied electron transfer from TMADH to ETF, investigated the reduction potentials of the bound ETF cofactor, and analyzed complex formation. We show that mutation of Arg-alpha237 substantially destabilizes the semiquinone couple of the bound FAD and impedes electron transfer from TMADH to ETF. Crystallographic structures of the mutant ETF proteins indicate that mutation does not perturb the overall structure of ETF, but leads to disruption of an electrostatic network at an ETF domain boundary that likely affects the dynamic properties of ETF in the crystal and in solution. We show that Arg-alpha237 is required for TMADH to structurally imprint the as-purified semiquinone form of wild-type ETF and that the ability of TMADH to facilitate this structural reorganization is lost following (i) redox cycling of ETF, or simple conversion to the oxidized form, and (ii) mutagenesis of Arg-alpha237. We discuss this result in light of recent apparent conflict in the literature relating to the structural imprinting of wild-type ETF. Our studies support a mechanism of electron transfer by conformational sampling as advanced from our previous analysis of the crystal structure of the TMADH-2ETF complex [Leys, D. , Basran, J. , Sutcliffe, M. J., and Scrutton, N. S. (2003) Nature Struct. Biol. 10, 219-225] and point to a key role for the Tyr-442 (TMADH) and Arg-alpha237 (ETF) residue pair in transiently stabilizing productive electron transfer configurations. Our work also points to the importance of Arg-alpha237 in controlling the thermodynamics of electron transfer, the dynamics of ETF, and the protection of reducing equivalents following disassembly of the TMADH-2ETF complex.

PubMed: 18407658
DOI: 10.1021/bi800127d

実験手法

X-RAY DIFFRACTION (1.9 Å)