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Galactoside-Binding Site in LacY

机译：金属糖苷绑定位点

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摘要

Although an X-ray crystal structure of lactose permease (LacY) has been presented with bound galactopyranoside, neither the sugar nor the residues ligating the sugar can be identified with precision at ～3.5 ?. Therefore, additional evidence is important for identifying side chains likely to be involved in binding. On the basis of a clue from site-directed alkylation suggesting that Asn272, Gly268, and Val264 on one face of helix VIII might participate in galactoside binding, molecular dynamics simulations were conducted initially. The simulations indicate that Asn272 (helix VIII) is sufficiently close to the galactopyranosyl ring of a docked lactose analogue to play an important role in binding, the backbone at Gly268 may be involved, and Val264 does not interact with the bound sugar. When the three side chains are subjected to site-directed mutagenesis, with the sole exception of mutant Asn272 → Gln, various other replacements for Asn272 either markedly decrease affinity for the substrate (i.e., high K_D) or abolish binding altogether. However, mutant Gly268 → Ala exhibits a moderate 8-fold decrease in affinity, and binding by mutant Val264 → Ala is affected only minimally. Thus, Asn272 and possibly Gly268 may comprise additional components of the galactoside-binding site in LacY.
机译：尽管已经用结合的半乳糖苷呈乳糖允许释放酶（花边）的X射线晶体结构，但是糖和残留物可以在〜3.5的〜3.5℃下鉴定糖的残留物。因此，额外的证据对于识别可能参与结合的侧链是重要的。基于来自位点导向的烷基化的线索，表明ASN272，GLY268和VAL264在Helix VIII的一面可能参与半乳糖糖苷结合，最初进行分子动力学模拟。该模拟表明ASN272（HELIX VIII）足够靠近对接乳糖类似物的半乳糖糖基环，以在结合中发挥重要作用，可以涉及GLY268的骨架，并且VAL264不会与结合的糖相互作用。当三侧链进行定点诱变时，突变体ASN272→GLN的唯一例外，ASN272的各种其他替代物明显降低对基材（即，高K_D）或完全废旧结合的亲和力。然而，突变颗粒颗粒→ALA表现出中等的8倍以下的亲和力减少，并且突变体Val264→Ala的结合仅对微小的影响。因此，ASN272和可能的GLY268可包括在花边中的半乳糖苷结合位点的另外的组分。

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  • 来源
    《Biochemistry》 |2014年第9期|共8页
  • 作者

    Xiaoxu Jiang; Maria Katerina R. Villafuerte; Magnus Andersson; Stephen H. White; H. Ronald Kaback;

  • 作者单位

    Department of Physiology and Department of Microbiology Immunology and Molecular Genetics Molecular Biology Institute University of California at Los Angeles Los Angeles California 90095-7327 United States;

    Department of Physiology and Department of Microbiology Immunology and Molecular Genetics Molecular Biology Institute University of California at Los Angeles Los Angeles California 90095-7327 United States;

    Department of Physiology and Biophysics and Center for Biomembrane Systems University of California at Irvine Irvine California 92697-4560 United States;

    Department of Physiology and Biophysics and Center for Biomembrane Systems University of California at Irvine Irvine California 92697-4560 United States;

    Department of Physiology and Department of Microbiology Immunology and Molecular Genetics Molecular Biology Institute University of California at Los Angeles Los Angeles California 90095-7327 United States;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    LacY; Asn272; Gly268;

    机译：花边;ASN272;GLY268;

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