Zoghbi Maria E, Nouel Barreto Annabella, Hernandez Alex L
Department of Molecular Cell Biology, School of Natural Sciences, University of California Merced, Merced, California; Health Sciences Research Institute, University of California Merced, Merced, California.
Quantitative Systems Biology Graduate Program, University of California Merced, Merced, California.
Biophys J. 2025 Apr 1;124(7):1117-1131. doi: 10.1016/j.bpj.2025.02.016. Epub 2025 Feb 18.
Humans have three known ATP-binding cassette (ABC) transporters in the inner mitochondrial membrane (ABCB7, ABCB8, and ABCB10). ABCB10, the most studied of them thus far, is essential for normal red blood cell development and protection against oxidative stress, and it was recently found to export biliverdin, a heme degradation product with antioxidant properties. The molecular mechanism underlying the function of ABC transporters remains controversial. Their nucleotide binding domains (NBDs) must dimerize to hydrolyze ATP, but capturing the transporters in such conformation for structural studies has been experimentally difficult, especially for ABCB10 and related eukaryotic transporters. Purified transporters are commonly studied in detergent micelles, or after their reconstitution in nanodiscs, usually at nonphysiological temperature and using nonhydrolyzable ATP analogs or mutations that prevent ATP hydrolysis. Here, we have used luminescence resonance energy transfer to evaluate the effect of experimental conditions on the NBD dimerization of ABCB10. Our results indicate that all conditions used for determination of currently available ABCB10 structures have failed to induce NBD dimerization. ABCB10 in detergent responded only to MgATP at 37°C, whereas reconstituted protein shifted toward dimeric NBDs more easily, including in response to MgAMP-PNP and even present NBD dimerization with MgATP at room temperature. The nanodisc's size affects the nucleotide-free conformational equilibrium of ABCB10 and the response to ATP in the absence of magnesium, but for all analyzed sizes (scaffold proteins MSP1D1, MSP1E3D1, and MSP2N2), a conformation with dimeric NBDs is clearly preferred during active ATP hydrolysis (MgATP, 37°C). These results highlight the sensitivity of this human ABC transporter to experimental conditions and the need for a more cautious interpretation of structural models obtained under far from physiological conditions. A dimeric NBD conformation that has been elusive in previous studies seems to be dominant during MgATP hydrolysis at physiological temperature.
人类线粒体内膜中有三种已知的ATP结合盒（ABC）转运蛋白（ABCB7、ABCB8和ABCB10）。ABCB10是迄今为止研究最多的一种，对正常红细胞发育和抗氧化应激至关重要，最近发现它能输出胆绿素，一种具有抗氧化特性的血红素降解产物。ABC转运蛋白功能的分子机制仍存在争议。它们的核苷酸结合结构域（NBDs）必须二聚化才能水解ATP，但在结构研究中捕获处于这种构象的转运蛋白在实验上很困难，尤其是对于ABCB10和相关的真核转运蛋白。纯化的转运蛋白通常在去污剂胶束中进行研究，或者在它们重构到纳米盘之后进行研究，通常是在非生理温度下，并使用不可水解的ATP类似物或阻止ATP水解的突变体。在这里，我们利用发光共振能量转移来评估实验条件对ABCB10的NBD二聚化的影响。我们的结果表明，用于确定目前可用的ABCB10结构的所有条件都未能诱导NBD二聚化。去污剂中的ABCB10仅在3°C时对MgATP有反应，而重构的蛋白更容易向二聚化的NBDs转变，包括对MgAMP-PNP的反应，甚至在室温下与MgATP一起出现NBD二聚化。纳米盘的大小会影响ABCB10的无核苷酸构象平衡以及在没有镁的情况下对ATP的反应，但对于所有分析的大小（支架蛋白MSP1D1、MSP1E3D1和MSP2N2），在活跃的ATP水解（MgATP，37°C）过程中，明显更倾向于二聚化NBDs的构象。这些结果突出了这种人类ABC转运蛋白对实验条件的敏感性，以及对在远离生理条件下获得的结构模型进行更谨慎解释的必要性。在先前研究中难以捉摸的二聚化NBD构象在生理温度下的MgATP水解过程中似乎占主导地位。
