Paulino Cristina, Kühlbrandt Werner
Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
Elife. 2014 Jan 28;3:e01412. doi: 10.7554/eLife.01412.
We examined substrate-induced conformational changes in MjNhaP1, an archaeal electroneutral Na(+)/H(+)-antiporter resembling the human antiporter NHE1, by electron crystallography of 2D crystals in a range of physiological pH and Na(+) conditions. In the absence of sodium, changes in pH had no major effect. By contrast, changes in Na(+) concentration caused a marked conformational change that was largely pH-independent. Crystallographically determined, apparent dissociation constants indicated ∼10-fold stronger Na(+) binding at pH 8 than at pH 4, consistent with substrate competition for a common ion-binding site. Projection difference maps indicated helix movements by about 2 Å in the 6-helix bundle region of MjNhaP1 that is thought to contain the ion translocation site. We propose that these movements convert the antiporter from the proton-bound, outward-open state to the Na(+)-bound, inward-open state. Oscillation between the two states would result in rapid Na(+)/H(+) antiport. DOI: http://dx.doi.org/10.7554/eLife.01412.001.
我们通过在一系列生理pH值和Na⁺条件下对二维晶体进行电子晶体学研究，考察了MjNhaP1（一种类似于人类转运体NHE1的古细菌电中性Na⁺/H⁺反向转运体）中底物诱导的构象变化。在没有钠的情况下，pH值的变化没有显著影响。相比之下，Na⁺浓度的变化引起了明显的构象变化，且这种变化在很大程度上与pH值无关。晶体学确定的表观解离常数表明，在pH 8时Na⁺结合力比pH 4时强约10倍，这与底物竞争共同离子结合位点一致。投影差异图表明，MjNhaP1的6螺旋束区域（被认为包含离子转运位点）的螺旋移动了约2 Å。我们认为这些移动将反向转运体从质子结合的外向开放状态转变为Na⁺结合的内向开放状态。两种状态之间的振荡将导致快速的Na⁺/H⁺反向转运。DOI: http://dx.doi.org/10.7554/eLife.01412.001 。
