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首页> 外文期刊>Journal of Molecular Biology >Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding.
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Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding.

机译：通过NMR研究的低分子量蛋白质酪氨酸磷酸酶在单体二聚体平衡中的分子内动力学：靶结合后动力学变化的模型。

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摘要

Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1). 1H and 15N NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. 15N relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer.
机译：低分子量蛋白酪氨酸磷酸酶（LMW-PTP）在溶液中以磷酸结合状态二聚，解离常数K（d）= 1.5（+/- 0.1）mM，解离速率约为10（4） s（-1）。 1H和15N NMR化学位移确定了二聚体界面，这与在二聚体S19A突变体的晶体结构中观察到的非常吻合。每个分子的两个酪氨酸残基与另一个分子的活性位点相互作用，这意味着该二聚体可以作为LMW-PTP与靶蛋白之间复合物的模型。从在四种不同蛋白质浓度下获得的弛豫数据推断出单体和二聚体态的15N弛豫率。提取了令人满意的精度的单体弛豫数据，从而可以对皮秒至纳秒级的骨干波动进行无模型分析。由于外推误差和较高浓度下可能存在高阶低聚物，因此二聚体弛豫数据的质量较低。定性比较单体和表观二聚体状态下的阶跃参数表明，形成二聚体界面的环在二聚化后变得刚性。单体-二聚体交换和分子内构象交换的定性信息是从自相关和交叉相关弛豫速率的浓度依赖性中获得的。含有重要催化作用的Asp129的环在单体和二聚体（目标结合）状态的不同构象之间波动。交换速率与催化反应步骤的交换速率相当好，支持了催化和酶动力学可能耦合的现有假设。 Trp49的侧链对底物的特异性很重要，它在单体中显示出构象动力学，该构象动力学在二聚体形成时被大量淬灭，这表明结合与单个侧链构象子的选择有关。

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