Karamyshev A L, Karamysheva Z N, Kajava A V, Ksenzenko V N, Nesmeyanova M A
Research group "Protein Secretion in Bacteria", Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia.
J Mol Biol. 1998 Apr 10;277(4):859-70. doi: 10.1006/jmbi.1997.1617.
A wide range (69) of mutant Escherichia coli alkaline phosphatases with single amino acid substitutions at positions from -5 to +1 of the signal peptide were obtained for studying protein processing as a function of the primary structure of the cleavage region. Amber suppressor mutagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (ii) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These data give new experimental support for the "-3, -1 rule". Only Ala, Gly and Ser at position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conservative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organisms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisingly, efficient processing was provided mainly by large amino acid residues at position -2 and by middle-sized residues at position -5, indicating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data suggests an extended beta-conformation of the -5 to -1 region in the signal peptidase binding pocket.
为了研究蛋白质加工过程与切割区域一级结构的关系，我们获得了一系列（69种）信号肽从-5到+1位置有单个氨基酸替换的突变型大肠杆菌碱性磷酸酶。用于创建突变蛋白的琥珀抑制突变包括：（i）在phoA基因的相应位置引入琥珀突变；（ii）在产生对丙氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、亮氨酸、赖氨酸、苯丙氨酸、脯氨酸、丝氨酸和酪氨酸具有特异性的琥珀抑制tRNA的大肠杆菌菌株中表达每个突变的phoA等位基因。-3和-1位置的大多数氨基酸替换导致蛋白质加工完全受阻。这些数据为“-3，-1规则”提供了新的实验支持。只有-1位置的丙氨酸、甘氨酸和丝氨酸允许蛋白质加工，其中丙氨酸的加工速率最高。结果表明，与真核生物相比，革兰氏阴性菌信号肽-1位置的氨基酸性质更为保守。-3位置不太规则，因为该位置不仅丙氨酸、丝氨酸和甘氨酸，而且亮氨酸和半胱氨酸也允许加工。-4位置的突变对加工影响不大。令人惊讶的是，高效加工主要由-2位置的大氨基酸残基和-5位置的中等大小残基提供，这表明加工速率不仅受-1和-3位置氨基酸残基大小的影响。结合突变和统计数据对切割位点进行的构象分析表明，信号肽酶结合口袋中-5到-1区域呈延伸的β构象。
