Alasadi Eman A, Choi Wonseok, Chen Xiaobing, Cotruvo Joseph A, Baiz Carlos R
Department of Chemistry, University of Texas at Austin, 105 E 24th St. A5300, Austin, Texas 78712, United States.
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
ACS Chem Biol. 2024 May 17;19(5):1056-1065. doi: 10.1021/acschembio.3c00789. Epub 2024 Apr 15.
Lanmodulins are small, ∼110-residue proteins with four EF-hand motifs that demonstrate a picomolar affinity for lanthanide ions, making them efficient in the recovery and separation of these technologically important metals. In this study, we examine the thermodynamic and structural complexities of lanthanide ion binding to a 41-residue domain, EF 2-3, that constitutes the two highest-affinity metal-binding sites in the lanmodulin protein from . Using a combination of circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we characterize the metal binding capabilities of EF 2-3. ITC demonstrates that binding occurs between peptide and lanthanides with conditional dissociation constants () in the range 20-30 μM, with no significant differences in the values for La, Eu, and Tb at pH 7.4. In addition, CD spectroscopy suggests that only one binding site of EF 2-3 undergoes a significant conformational change in the presence of lanthanides. 2D IR spectroscopy demonstrates the presence of both mono- and bidentate binding configurations in EF 2-3 with all three lanthanides. MD simulations, supported by Eu luminescence measurements, explore these results, suggesting a competition between water-lanthanide and carboxylate-lanthanide interactions in the EF 2-3 domain. These results underscore the role of the core helical bundle of the protein architecture in influencing binding affinities and communication between the metal-binding sites in the full-length protein.
镧调蛋白是一种小蛋白，约含110个氨基酸残基，具有四个EF手基序，对镧系离子表现出皮摩尔亲和力，使其在回收和分离这些具有重要技术意义的金属方面效率很高。在本研究中，我们研究了镧系离子与一个由41个氨基酸残基组成的结构域EF 2-3结合的热力学和结构复杂性，该结构域构成了来自[具体来源未提及]的镧调蛋白中两个亲和力最高的金属结合位点。我们结合使用圆二色性（CD）光谱、等温滴定量热法（ITC）、二维红外（2D IR）光谱和分子动力学（MD）模拟，对EF 2-3的金属结合能力进行了表征。ITC表明，肽与镧系元素之间发生结合，条件解离常数（）在20-30 μM范围内，在pH 7.4时，La、Eu和Tb的 值没有显著差异。此外，CD光谱表明，在镧系元素存在的情况下，EF 2-3只有一个结合位点发生了显著的构象变化。2D IR光谱表明，EF 2-3与所有三种镧系元素都存在单齿和双齿结合构型。由Eu发光测量支持的MD模拟对这些结果进行了探究，表明在EF 2-3结构域中，水-镧系元素和羧酸盐-镧系元素相互作用之间存在竞争。这些结果强调了蛋白质结构的核心螺旋束在影响全长蛋白质中金属结合位点的结合亲和力和通讯方面的作用。
