Panchal R G, Bayley H
Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.
J Biol Chem. 1995 Sep 29;270(39):23072-6. doi: 10.1074/jbc.270.39.23072.
alpha-Hemolysin (alpha HL), a pore-forming polypeptide of 293 amino acids, is secreted by Staphylococcus aureus as a water-soluble monomer. Residues that play key roles in the formation of functional heptameric pores on rabbit red blood cells (rRBC) have been identified previously by site-directed mutagenesis. alpha HL-H35N, in which the histidine at position 35 of the wild-type sequence is replaced with asparagine, is nonlytic and is arrested in assembly as a heptameric prepore. In this study, second-site revertants of H35N that have the ability to lyse rRBC were generated by error-prone PCR under conditions designed to produce single base changes. The analysis of 22 revertants revealed new codons clustered predominantly in three distinct regions of the H35N gene. One cluster includes amino acids 107-111 (four revertants) and another residues 144-155 (five revertants). These two clusters flank the central glycine-rich loop of alpha HL, which previously has been implicated in formation of the transmembrane channel, and encompass residues Lys-110 and Asp-152 that, like His-35, are crucial for lytic activity. The third cluster lies in the region spanning amino acids 217-228 (eight revertants), a region previously unexplored by mutagenesis. Single revertants were found at amino acid positions 84 and 169. When compared with H35N, the heptameric prepores formed by the revertants underwent more rapid conversion to fully assembled pores, as determined by conformational analysis by limited proteolysis. The rate of conversion to the fully assembled pore was strongly correlated with hemolytic activity. Previous work has suggested that the N terminus of alpha HL and the central loop cooperate in the final step of assembly. The present study suggests that the key N-terminal residue His-35 operates in conjunction with residues flanking the loop and C-terminal residues in the region 217-228. Hence, reversion mutagenesis extends the linear analysis that has been provided by direct point mutagenesis.
α-溶血素（αHL）是一种由293个氨基酸组成的成孔多肽，由金黄色葡萄球菌作为水溶性单体分泌。先前已通过定点诱变鉴定出在兔红细胞（rRBC）上功能性七聚体孔形成中起关键作用的残基。αHL-H35N中，野生型序列第35位的组氨酸被天冬酰胺取代，它不具有溶细胞作用，并且在组装成七聚体前体孔时停滞。在本研究中，通过易错PCR在设计用于产生单碱基变化的条件下产生了具有裂解rRBC能力的H35N的第二位点回复突变体。对22个回复突变体的分析揭示了新密码子主要聚集在H35N基因的三个不同区域。一个簇包括氨基酸107 - 111（四个回复突变体），另一个簇包括残基144 - 155（五个回复突变体）。这两个簇位于αHL富含甘氨酸的中央环两侧，该中央环先前被认为与跨膜通道的形成有关，并且包含与His-35一样对裂解活性至关重要的Lys-110和Asp-152残基。第三个簇位于跨越氨基酸217 - 228的区域（八个回复突变体），这是一个先前未通过诱变探索的区域。在氨基酸位置84和169发现了单个回复突变体。通过有限蛋白酶解的构象分析确定，与H35N相比，回复突变体形成的七聚体前体孔向完全组装孔的转化更快。向完全组装孔的转化速率与溶血活性密切相关。先前的研究表明，αHL的N末端和中央环在组装的最后一步协同作用。本研究表明，关键的N末端残基His-35与环两侧的残基以及区域217 - 228中的C末端残基共同起作用。因此，回复突变诱变扩展了直接点诱变所提供的线性分析。
