PDF(Pubmed)
Abstract：
SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.
摘要：
SlyD是一种广泛存在的原核FKBP家族脯氨酸酰异构酶，具有额外的伴侣结构域。通常,例如在大肠杆菌中，在其C末端发现第三结构域,其结合镍并为镍酶生物发生提供。已发现SlyD结合通过Tat途径易位的蛋白质的信号肽，折叠蛋白质跨膜转运的系统。使用肽阵列来分析这些信号肽的相互作用，我们发现SlyD只与带正电荷的肽相互作用，优先于精氨酸而不是赖氨酸，和大的疏水残基增强结合。特别是双精氨酸基序被识别，与疏水残基相邻的一对高度保守的精氨酸。使用等温滴定量热法（ITC），纯化的SlyD和含信号肽的模型Tat底物，我们可以证明，野生型双精氨酸信号肽的结合亲和力高于RR>KK突变的变体，确认SlyD识别正电荷，精氨酸优先于赖氨酸。通过突变SlyD变体的ITC进一步分析了伴侣结构域表面的负电荷和伴侣活性位点中的疏水残基的特定作用。我们的数据表明，活性位点的假定关键疏水残基对于结合确实至关重要，并且该结合受到伴侣结构域上的负电荷的影响。正电荷的识别可能通过伴侣域的大的带负电荷的表面区域来实现，这是高度保守的，尽管个别位置是可变的。
