Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA; Natural Science Division, Campbellsville University, Campbellsville, KY 42718, USA.
Biochim Biophys Acta Biomembr. 2021 Dec 1;1863(12):183771. doi: 10.1016/j.bbamem.2021.183771. Epub 2021 Sep 7.
The bacteriophage infection cycle is terminated at a predefined time to release the progeny virions via a robust lytic system composed of holin, endolysin, and spanin proteins. Holin is the timekeeper of this process. Pinholin S is a prototype holin of phage Φ21, which determines the timing of host cell lysis through the coordinated efforts of pinholin and antipinholin. However, mutations in pinholin and antipinholin play a significant role in modulating the timing of lysis depending on adverse or favorable growth conditions. Earlier studies have shown that single point mutations of pinholin S alter the cell lysis timing, a proxy for pinholin function as lysis is also dependent on other lytic proteins. In this study, continuous wave electron paramagnetic resonance (CW-EPR) power saturation and double electron-electron resonance (DEER) spectroscopic techniques were used to directly probe the effects of mutations on the structure and conformational changes of pinholin S that correlate with pinholin function. DEER and CW-EPR power saturation data clearly demonstrate that increased hydrophilicity induced by residue mutations accelerate the externalization of antipinholin transmembrane domain 1 (TMD1), while increased hydrophobicity prevents the externalization of TMD1. This altered hydrophobicity is potentially accelerating or delaying the activation of pinholin S. It was also found that mutations can influence intra- or intermolecular interactions in this system, which contribute to the activation of pinholin and modulate the cell lysis timing. This could be a novel approach to analyze the mutational effects on other holin systems, as well as any other membrane protein in which mutation directly leads to structural and conformational changes.
噬菌体感染周期在预定时间结束，通过由溶菌素、内溶素和间隔蛋白组成的强大裂解系统释放子代病毒粒子。溶菌素是这个过程的定时器。Pinholin S 是噬菌体 Φ21 的原型溶菌素，通过 pinholin 和 antipinholin 的协同作用，决定宿主细胞裂解的时间。然而，pinholin 和 antipinholin 的突变在调节裂解时间方面起着重要作用，这取决于不利或有利的生长条件。早期的研究表明，pinholin S 的单点突变会改变细胞裂解时间，这是裂解功能的一个替代指标，因为裂解也依赖于其他裂解蛋白。在这项研究中，连续波电子顺磁共振（CW-EPR）功率饱和和双电子-电子共振（DEER）光谱技术被用来直接探测突变对 pinholin S 结构和构象变化的影响，这些变化与 pinholin 功能相关。DEER 和 CW-EPR 功率饱和数据清楚地表明，残基突变诱导的亲水性增加加速了 antipinholin 跨膜域 1（TMD1）的外向化，而疏水性增加则阻止了 TMD1 的外向化。这种改变的疏水性可能加速或延迟 pinholin S 的激活。还发现突变可以影响这个系统中的分子内或分子间相互作用，这有助于 pinholin 的激活，并调节细胞裂解时间。这可能是分析其他溶菌素系统以及任何其他突变直接导致结构和构象变化的膜蛋白突变效应的一种新方法。
