Choi S K, Ljungdahl L G
Department of Biochemistry and Molecular Biology, Center for Biological Resources Recovery, University of Georgia, Athens, 30602-7229, USA.
Biochemistry. 1996 Apr 16;35(15):4906-10. doi: 10.1021/bi9524631.
The cellulosome of Clostridium thermocellum is a multipolypeptide complex of structural and catalytic subunits. Several of the catalytic subunits have at the carboxyl end a conserved duplicated region (CDR) which interacts with internally repeated elements (IREs) of scaffolding subunits such as CipA. This interaction requires calcium. The two parts of the CDR region here designated CDR1 and CDR2 (closest to the carboxyl end) each consist of about 20 amino acids residues. As shown in our previous paper [Choi, S.K., & Ljungdahl, L.G. (1996) Biochemistry 35, 4897-4905], treatment of the cellulosome with ethylenediaminetetraacetic acid (EDTA) under aerobic conditions disintegrates the cellulosome with formation of truncated catalytic subunits. The cleavage is at a specific asparagine residue located within CDR1 and occurs with complete loss of CDR2. Two branched peptides containing the amino acid sequences of CDR1 and CDR2 (designated bCDR1 and bCDR2) were synthesized, and specific antibodies were raised against them. These antibodies did not cross react with bCDR1 or bCDR2, respectively. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, it was observed that about 15 subunits of the cellulosome reacted with anti-bCDR1 and anti-bCDR2. In a similar experiment with EDTA-treated cellulosomes, these subunits reacted with anti-bCDR1 but not with anti-bCDR2, showing that they lost the bCDR2 epitope and were truncated. The peptide bCDR1 binds calcium, whereas bCDR2 does not. Furthermore, bCDR1 but not bCDR2 binds to CipA, presumably at IRE regions. This binding requires calcium. A model is proposed for the binding of the catalytic subunits to CipA which involves CDR1, an IRE, and calcium.
嗜热栖热梭菌的纤维小体是一种由结构亚基和催化亚基组成的多聚体复合物。其中几个催化亚基在羧基末端有一个保守的重复区域（CDR），该区域与支架亚基（如CipA）的内部重复元件（IRE）相互作用。这种相互作用需要钙。这里将CDR区域的两部分分别命名为CDR1和CDR2（最靠近羧基末端），每部分都由大约20个氨基酸残基组成。正如我们之前的论文[Choi, S.K., & Ljungdahl, L.G. (1996) Biochemistry 35, 4897 - 4905]所示，在有氧条件下用乙二胺四乙酸（EDTA）处理纤维小体，会使纤维小体解体并形成截短的催化亚基。切割发生在CDR1内的一个特定天冬酰胺残基处，并且CDR2会完全丢失。合成了包含CDR1和CDR2氨基酸序列的两条分支肽（分别命名为bCDR1和bCDR2），并针对它们制备了特异性抗体。这些抗体分别与bCDR1或bCDR2不发生交叉反应。经过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和蛋白质印迹分析后，观察到纤维小体中约15个亚基与抗bCDR1和抗bCDR2发生反应。在对经EDTA处理的纤维小体进行的类似实验中，这些亚基与抗bCDR1发生反应，但不与抗bCDR2发生反应，表明它们失去了bCDR2表位并且被截短了。肽bCDR1能结合钙，而bCDR2不能。此外，bCDR1能与CipA结合，推测是在IRE区域，这种结合需要钙。提出了一个催化亚基与CipA结合的模型，该模型涉及CDR1、一个IRE和钙。
