Surface loop motion in FepA.
Surface loop motion in FepA.
复制标题
FepA 中的表面循环运动。
DOI：
10.1128/jb.184.17.4906-4911.2002
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发表时间：
2002
影响因子：
3.2
通讯作者：
Klebba,PhillipE
中科院分区：
文献类型：
--
作者：
Scott,DanielC;Newton,SaleteMC;Klebba,PhillipE
使用赖氨酸特异性可切割的交联剂乙二醇双（磺基琥珀酰亚胺基琥珀酸酯）（Sulfo-EGS），我们研究了大肠杆菌FepA在其运输铁载体铁肠杆菌素的过程中表面环的构象运动。定点诱变确定Sulfo-EGS与两个赖氨酸K332和K483以及外膜蛋白表面环中至少两个其他未鉴定的赖氨酸残基反应。该试剂将FepA L7中的K483与L5中的K332交联，形成我们指定为条带1的产物，或与主要外膜蛋白OmpF、OmpC和OmpA交联，形成条带2。铁肠杆菌素与FepA的结合不能阻止Sulfo-EGS修饰K483，但能阻断其与OmpF/C和OmpA的交联，并减少其与K332的偶联。这些数据表明，FepA的环在体内经历构象变化，从无配体的开放状态到配体结合的闭合状态，变化幅度约为15 A/cm。FepA L7与OmpF、OmpC或OmpA的偶联是TonB独立的，不受解偶联剂CCCP（羰基氰化物-氯苯腙）和DNP（2，4-二硝基苯酚）的影响，但完全被氰化物抑制。
Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops ofEscherichia coliFepA during its transport of the siderophore ferric enterobactin. Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein. The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2. Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332. These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 Å, from a ligand-free open state to a ligand-bound closed state. The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanidem-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.