Effects of Mutations and Ligands on the Thermostability of the L-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human L-Type Amino Acid Transporters
Effects of Mutations and Ligands on the Thermostability of the L-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human L-Type Amino Acid Transporters
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DOI：
10.3390/ijms19030918
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发表时间：
2018-03-01
影响因子：
5.6
通讯作者：
Fotiadis, Dimitrios
中科院分区：
文献类型：
--
作者：
Ilgu, Huseyin;Jeckelmann, Jean-Marc;Fotiadis, Dimitrios
l -精氨酸/agmatine转运体AdiC是SLC7家族的原核成员，它使致病性肠杆菌能够在极酸性的胃环境中生存。从大肠杆菌中分离出的野生型AdiC及其先前报道的点突变体N22A和S26A均同源过表达并纯化至同源性。采用基于尺寸排除色谱法的热稳定性测定法，测定了在l -精氨酸（Arg）、精氨酸、l -精氨酸甲酯和l -精氨酸酰胺等配体存在或不存在的情况下，纯化的AdiC变体的熔化温度（T(m)s）。得到的T(m)s表明AdiC变异在配体结合后趋于稳定，其中T(m)s与配体结合亲和力呈正相关。考虑到本研究和之前的研究结果，我们重新审视了AdiC残基S26在精氨酸结合中的作用，并提出了精氨酸的a-羧酸基与AdiC主链的酰胺基之间的相互作用。在人类SLC7家族成员l型氨基酸转运体-1 （LAT1; SLC7A5）底物结合的背景下，基于同源性建模和氨基酸序列分析，讨论了LAT1中S66与AdiC中S26的类似作用。最后，我们提出了l -氨基酸底物与SLC7家族LATs的结合机制。
The L-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (T(m)s) of the purified AdiC variants in the absence and presence of the selected ligands L-arginine (Arg), agmatine, L-arginine methyl ester, and L-arginine amide. The resulting T(m)s indicated stabilization of AdiC variants upon ligand binding, in which T(m)s and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the a-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member L-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for L-amino acid substrates to LATs from the SLC7 family.