STRUCTURAL AND FUNCTIONAL DOMAINS OF THE LARGE SUBUNIT OF THE BACTERIOPHAGE-T3 DNA PACKAGING ENZYME - IMPORTANCE OF THE C-TERMINAL REGION IN PROHEAD BINDING
STRUCTURAL AND FUNCTIONAL DOMAINS OF THE LARGE SUBUNIT OF THE BACTERIOPHAGE-T3 DNA PACKAGING ENZYME - IMPORTANCE OF THE C-TERMINAL REGION IN PROHEAD BINDING
复制标题
DOI：
10.1006/jmbi.1994.0052
复制
发表时间：
1995-02-03
影响因子：
5.6
通讯作者：
FUJISAWA, H
中科院分区：
文献类型：
--
作者：
MORITA, M;TASAKA, M;FUJISAWA, H
在噬菌体T3的头部组装过程中，DNA在非衣壳包装蛋白（基因18和19的产物（gp 18和gp 19））的帮助下被包装到预先形成的蛋白质外壳中，称为前头部。我们已经建立了一个确定的系统，由纯化的gp 18，gp 19和proheads组成的T3 DNA的体外包装。我们以前的研究结果使用定义的体外系统表明，在DNA包装的顺序事件：包装蛋白，gp 18和gp 19，分别结合DNA和prohead，这些复合物缔合形成直接的前体复合物的DNA易位到头部。前体复合物的形成需要ATP作为变构效应物。随后的DNA易位由ATP水解驱动。gp 19是一种ATP结合蛋白，通过与ATP相互作用在DNA包装中发挥多种作用。gp 19通过与ATP结合而改变其构象，如从有限蛋白水解的分析所判断的。有限的蛋白水解切割的网站进行了测定和定位的gp 19多肽（586个氨基酸残基）的图像由ATP诱导的gp 19的构象变化。胰蛋白酶消化产生的C-末端片段结合的prohead和抑制DNA包装的完整的gp 19在竞争的方式。另一方面，N-末端片段不结合前体，也不抑制DNA包装。这些结果定义了在gp 19的C末端的前头部结合结构域。为了更精确地鉴定前头部结合结构域，构建了缺失gp 19末端C末端的最后10个和15个氨基酸的缺失突变体（分别为gp 19-Delta C10和gp 19-Delta C15）。这些突变体蛋白在ATP存在或不存在下的有限胰蛋白酶消化模式基本上与gp 19-wt相同，表明这些缺失不会改变构象及其ATP响应。gp 19-Delta C15缺乏前头部结合活性，因此，DNA包装活性gp 19-Delta C10具有显著的DNA包装活性，尽管它降低到gp 19-wt的十分之一。这些结果表明，残基L571至D576的gp 19的C-末端区域是至关重要的prohead的结合和最后10个残基D577至W586的C-末端似乎是重要的稳定结合的gp 19的prohead。
During head assembly of phage T3, DNA is packaged into a preformed protein shell, called the prohead, with the aid of non-capsid packaging proteins, the products of genes 18 and 19 (gp18 and gp19). We have developed a defined system, composed of purified gp18, gp19 and proheads for in vitro packaging of T3 DNA. Our previous results using the defined in vitro system indicate the sequential events in DNA packaging: the packaging proteins, gp18 and gp19, bind DNA and proheads, respectively These complexes associate to form a direct precursor complexes for DNA translocation into the head. The formation of the precursor complexes requires ATP as an allosteric effector. Subsequent DNA translocation is driven by ATP hydrolysis. gp19 is an ATP binding protein that plays multiple roles in DNA packaging through interaction with ATP. gp19 changes its conformation by binding to ATP, as judged from the analysis of limited proteolysis. Sites cleaved by limited proteolysis were determined and mapped on the gp19 polypeptide (586 amino acid residues) to image the conformational change of gp19 induced by ATP. C-Terminal fragments generated by trypsin digestion bound the prohead and inhibited DNA packaging by intact gp19 in a competitive manner. On the other hand, N-terminal fragments did not bind the prohead nor did they inhibit DNA packaging. These results define a prohead binding domain at the C terminus of gp19. To identify the prohead binding domain more precisely, deletion mutants lacking the last 10 and 15 amino acids (gp19-Delta C10 and gp19-Delta C15, respectively) of the extreme C terminus of gp19 were constructed. Limited tryptic digestion patterns of these mutant proteins in the presence or absence of ATP were basically the same as those of gp19-wt, indicating that the conformation and its ATP response were not changed by these deletions. gp19-Delta C15 lacked prohead binding activity and, therefore, DNA packaging activity gp19-Delta C10 had significant DNA packaging activity although it was reduced to one-tenth of that of gp19-wt. These results indicate that a C-terminal region of residues L571 to D576 of gp19 is crucial for prohead binding and that the last ten residues D577 to W586 of the C terminus seems to be important in stable binding of gp19 to the prohead.