Induced fit substrate binding to an archeal glutamate transporter homologue
Induced fit substrate binding to an archeal glutamate transporter homologue
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DOI：
10.1073/pnas.1300772110
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发表时间：
2013-07-23
影响因子：
11.1
通讯作者：
Fahlke, Christoph
中科院分区：
文献类型：
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作者：
Ewers, David;Becher, Toni;Fahlke, Christoph
兴奋性氨基酸转运体(EAATs)是一类终止哺乳动物中枢谷氨酸能突触传递的谷氨酸转运体。GLT(Ph)是Horikoshii的EAAT同源物，是目前已知的唯一具有三维结构的成员。在这里，我们研究了单一色氨酸突变体(L130W)GLT(Ph)在洗涤剂胶束中与底物结合的动力学。在低钠离子浓度下，L-天冬氨酸的加入导致W130在几十秒内的荧光变化呈现复杂的时间过程。随着[Na+]的增加，动力学以快组分[k(OBS，FAST)；K-D(Na+)=22+/-3 mm，n(Hill)=1.7+/-0.3]为主，随着[L-天冬氨酸]的增加，k(OBS，FAST)以饱和的方式增加到接近500OBS(-1)(6℃时)。L-天冬氨酸的结合动力学不同于L-半胱氨酸亚磺酸和D-天冬氨酸的结合动力学。L-半胱氨酸与天冬氨酸的结合亲和力高于L-天冬氨酸，但饱和速率较低，而D-天冬氨酸结合后的饱和速率较高。因此，在两个Na+与空转运蛋白结合后，GLT(Ph)通过诱导FIT结合氨基酸。交联和蛋白质降解实验表明，诱导FIT是由于螺旋发夹2的关闭造成的。GLT(Ph)的这种构象变化比大多数哺乳动物同系物快，而氨基酸结合率相似。我们的数据揭示了诱导FIT在EAAT底物选择中的重要性，并说明了这类转运蛋白如何同时完成高亲和力结合和谷氨酸的有效运输。
Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. Glt(Ph), an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) Glt(Ph) in detergent micelles. At low millimolar [Na+], the addition of L-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na+], the kinetics were dominated by a fast component [k(obs, fast); K-D (Na+) = 22 +/- 3 mM, n(Hill) = 1.7 +/- 0.3] with values of k(obs, fast) rising in a saturable manner to approximate to 500 s(-1) (at 6 degrees C) with increasing [L- aspartate]. The binding kinetics of L-aspartate differed from the binding kinetics of two alternative substrates: L-cysteine sulfinic acid and D-aspartate. L-cysteine sulfinic acid bound with higher affinity than L-aspartate but involved lower saturating rates, whereas the saturating rates after D-aspartate binding were higher. Thus, after the association of two Na+ to the empty transporter, Glt(Ph) binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for Glt(Ph) than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.