Spatial distribution of cytoplasmic domains of the Mg²⁺-transporter MgtE, in a solution lacking Mg²⁺, revealed by paramagnetic relaxation enhancement

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• 作者：Shunsuke Imai ; Tatsuro Maruyama ; Masanori Osawa ; Motoyuki Hattori ; Ryuichiro Ishitani ; Osamu Nureki ; Ichio Shimada
• 关键词：PRE ; paramagnetic relaxation enhancement ; TM ; transmembrane ; MgtECP ; cytoplasmic region of MgtE (residues 1&ndash ; 259) ; HEPES ; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ; MTSL ; S-(2 ; 2 ; 5 ; 5-tetramethyl-2 ; 5-dihydro-1H-pyrrol-3-yl)methyl methanesulfon
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2012
• 出版时间：October, 2012
• 年：2012
• 卷：1824
• 期：10
• 页码：1129-1135
• 全文大小：1302 K

文摘

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.