Stanley P, Koronakis V, Hardie K, Hughes C
Department of Pathology, University of Cambridge, UK.
Mol Microbiol. 1996 May;20(4):813-22. doi: 10.1111/j.1365-2958.1996.tb02519.x.
The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated. Each domain required 15-30 amino acids for basal recognition and 50-80 amino acids for wild-type acylation. The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca2+ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAII domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.
通过对原毒素与特定酰基转移酶HlyC相互作用的研究，进一步阐明了大肠杆菌溶血素及相关毒素激活（成熟）背后明显独特的脂肪酰化机制。在依赖于HlyC和酰基 - 酰基载体蛋白的体外成熟反应中，使用缺失的原毒素变体和原毒素肽作为底物，在1024个残基的原毒素proA上鉴定出两个独立的HlyC识别结构域，并且它们跨越了被脂肪酰化的两个靶赖氨酸残基K564（KI）和K690（KII）。每个结构域基础识别需要15 - 30个氨基酸，野生型酰化需要50 - 80个氨基酸。这两个结构域（FAI和FAII）在顺式和反式中相互竞争HlyC。FAI对HlyC的亲和力比FAII大约大四倍，导致在全毒素和肽衍生物中KI处的总体酰化率为80％，KII处的酰化率为20％。毒素成熟不需要其他proA序列，过量的Ca2 +会阻止两个赖氨酸的酰化。proA中FAI和FAII结构域之间以及相关原毒素上相应位点之间缺乏一级序列同一性，目前无法解释proA对HlyC识别的基础。
