肌动蛋白丝是由单个肌动蛋白构建的细胞中的动态蛋白质纤维。许多细胞功能，包括细胞运动，都是由恒定的灯丝组装和拆卸来调节的。拆卸阶段是由从灯丝内部释放磷酸基团开始的，但这个过程的细节几十年来一直困扰着科学家们。来自多特蒙德马克斯普朗克分子生理学研究所和法兰克福马克斯普朗克生物物理研究所的研究人员联手精确地确定了肌动蛋白中的一个区域，该区域充当磷酸盐退出的“分子后门”。使用各种技术，包括低温电子显微镜（cryo-EM）和分子动力学模拟，科学家们以前所未有的分子细节确定了肌动蛋白丝释放磷酸盐的机制。他们还描述了扭曲的后门如何能够更快地从与线虫性肌病（一种严重的肌肉疾病）相关的肌动蛋白突变体中释放磷酸盐。这项研究为进一步研究细胞中的动态肌动蛋白组装周期以及与肌动蛋白组织缺陷相关的疾病打开了大门。
The mysterious escape of phosphate
In eukaryotic cells, actin proteins join together (polymerize) into filaments that are part of the cell’s intricate supportive network, the cytoskeleton. The disassembly of old filaments is crucial for cell movement and is regulated by ATP hydrolysis — the reaction of ATP with water that cleaves a phosphate group and generates energy. Specifically, phosphate release from the filament core is the signal to the cell that the actin filament is old enough and can be dismantled into actin subunits. “The mechanism of phosphate release from actin filaments has remained enigmatic for decades,” says Wout Oosterheert, postdoc in the group of Stefan Raunser at the MPI Dortmund and first author of the publication.
The new results are built on previous research of Raunser’s group on actin that led to ground-breaking publications in 2015, 2018, and 2022 in the actin field. In the latter, the Raunser team determined high-resolution cryo-EM structures of actin filaments in three different states: bound to ATP, bound to ADP in the presence of the cleaved phosphate, and bound to ADP after release of the phosphate. However, in all structures, there was no opening or door in actin through which phosphate could escape from the filament. “Therefore, we surmised that there should be a backdoor that opens momentarily to release the phosphate, and then quickly closes again” says Raunser.
A multidisciplinary approach
MPI scientists have now tackled the problem from various angles. Since it was known that phosphate is released very rapidly from actin at the tip of the filament, called the barbed end, Raunser and his team determined its structure by cryo-EM. And indeed, only at the end of the filament, they found an open molecular backdoor, which explains the very fast phosphate release. However, it was still unclear how phosphate escapes from the actin subunits in the filament core. That’s where the expertise of Gerhard Hummer’s group from the MPI Frankfurt kicked in; they used the structural data from 2022 to perform molecular dynamics simulations and predict potential exit routes for the phosphate from the filament core. They then teamed up with the group of Peter Bieling (MPI Dortmund) to validate the possible routes by producing actin mutants potentially disrupting the molecular backdoor. They measured how fast they release the phosphate, and finally determined the high-resolution cryo-EM structures of the “fastest” candidates.
The mutational analysis revealed that the phosphate takes the same release route in the filament end and the filament core. The structures and interactions in the latter, however, need additional rearrangements that make it more difficult for the door to open. After phosphate cleavage, the backdoor remains predominantly closed (on average for 100 seconds) before opening for less than a second to let the phosphate leave. “This explains why we didn’t see an open backdoor arrangement in our cryo-EM data of 2022,” says Raunser.
The actin saga — To be continued…
One of the actin mutants analyzed, called N111S, is linked to the muscular disease nemaline myopathy and has therefore attracted the attention of the MPI scientists: the mutant always adopts an open backdoor and hence releases phosphate much faster than normal wild-type actin. “We propose that this ultrafast release may contribute to the pathophysiology in patients harboring this actin mutation,” says Oosterheert.
As a potential next step, the MPI scientists now want to uncover how phosphate release is controlled within the cell and what role the proteins that bind to actin play. In addition, their work now makes it possible to investigate other disease-related mutations in actin — an approach that may ultimately contribute to the development of new therapeutic strategies for these diseases.
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