Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution
Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution
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DOI：
10.1073/pnas.1816184116
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发表时间：
2019-03
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影响因子：
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通讯作者：
X. Ye;JiaBei Lin;L. Mayne;J. Shorter;S. Englander
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文献类型：
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作者：
X. Ye;JiaBei Lin;L. Mayne;J. Shorter;S. Englander
意义冷冻-EM革命现在提供了大型蛋白质分子机器结构的快照，但决定它们如何工作的基本机制和原理到目前为止还难以捉摸。所需要的是一种技术，当它们在生理溶液中活动时，可以研究它们的结构、结构动力学和能量学。本文利用氢交换质谱学方法对HSP104中的所有活性和非活性元素进行解析，表征了它们之间的相互作用和动力学，并对它们的能量学进行了独特的研究。结果从广义的结构和基于能量的角度揭示了HSP104是如何工作的。所使用的方法、所获得的结果以及它们所建议的能量方面的考虑将广泛适用于其他依赖ATPase的蛋白质机器。HSP104是一个大型的AAA+分子机器，它可以通过将底物蛋白质吸入其中央孔道来拯救困在无定形聚集体中的蛋白质和稳定的淀粉样蛋白。最近的低温电子显微镜研究了高分辨率的HSP104图像。我们使用氢交换质谱分析(HX-MS)来解析和表征HSP104的所有功能活性和非活性元素，其中许多元素是冷冻EM无法获得的。在全球水平上，HX MS证实了Hsp104六角体中的一个非规范的蛋白质间界面是冷冻-EM揭示的螺旋构象的标志，并测量了其在ATP水解下的快速构象循环。其他发现使人们能够重新解释监管中间领域的明显可变性。在详细的机制方面，HX MS确定了每个HSP104结构元件对不同结合的腺苷核苷酸(腺苷二磷酸、三磷酸腺苷、三磷酸腺苷和三磷酸腺苷γS)的反应。它们最敏感的区别是两个Walker A核苷酸结合片段。三磷酸腺苷类似物-三磷酸腺苷γS的绑定，紧密地重组了Walker A部分，并推动了全球从开放到关闭/延长的过渡。这种全球转变将部分三磷酸腺苷/三磷酸腺苷γ与S结合的能量携带到距离较远的中央孔道上。毛孔收缩，酪氨酸和其他与毛孔相关的环变得更加紧密，这似乎反映了需要能量的方向性拉力移位底物蛋白质。ATP水解为ADP允许Hsp104放松到其最低能量开放状态，准备重新开始循环。
Significance The cryo-EM revolution now provides snapshots of the structure of large protein molecular machines, but the fundamental mechanisms and principles that determine how they work have so far been elusive. What is needed is a technology that can investigate their architecture, structural dynamics, and energetics when they are active in physiological solution. This paper demonstrates the use of hydrogen exchange mass spectrometry methods to resolve all of the functionally active and inactive elements of Hsp104, characterize their interactions and dynamics, and uniquely their energetics. The results reveal in broad structural and energy-based terms how Hsp104 works. The methods used, the results obtained, and the energetic considerations that they suggest will be widely applicable to other ATPase-dependent protein machines. Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.