Significance of Individual Domains of ClpL: A Novel Chaperone from Streptococcus mutans.
Significance of Individual Domains of ClpL: A Novel Chaperone from Streptococcus mutans.
复制标题
DOI：
10.1021/acs.biochem.0c00544
复制
发表时间：
2020-09-15
期刊：
影响因子：
2.9
通讯作者：
Biswas I
中科院分区：
文献类型：
--
作者：
Jana B;Biswas I
ClpL是AAA+分子伴侣的HSP 100家族的成员，其广泛存在于革兰氏阳性细菌中，但令人惊讶地不存在于革兰氏阴性细菌中。ClpL参与各种细胞过程，包括胁迫耐受反应、长期存活、毒力和抗生素抗性。ClpL的特征很差，其分子伴侣活性的机制在很大程度上是不清楚的。在这里，我们对来自牙齿病原体变形链球菌的ClpL蛋白进行了生物化学表征，以了解其生物学功能。ClpL具有五个结构域：N-结构域、两个核苷酸结合结构域（NBD-1和NBD-2）、M-结构域和C-结构域。NBD-1和NBD-2分别含有用于ATP结合和水解的不同的步行者A和B基序。我们发现ClpL在溶液中主要以三聚体形式存在;然而，在ATP结合后，它迅速形成六聚体结构。为了研究结构-功能活性，我们构建了几个取代和缺失突变体。我们发现步行者A和B基序的突变干扰ATP水解和寡聚化。类似地，N-、M-和C-结构域的缺失消除了ATP酶活性和寡聚化。由于我们以前发现ClpL作为伴侣，我们分析了伴侣活性。令人惊讶的是，我们发现NBD-2突变体不显示任何伴侣活性，表明ATP结合和NBD-2的伴侣水解的必要性。然而，NBD-1突变体显示分子伴侣活性，但活性是可变的，这取决于突变的性质。我们的研究结果表明，与其他HSP 100家族分子伴侣不同，ClpL是一种新型分子伴侣，其活性不需要任何额外的二级分子伴侣。
ClpL is a member of the HSP100 family of the AAA+ chaperone that is widely present in Gram-positive but surprisingly absent in Gram-negative bacteria. ClpL is involved in various cellular processes including stress tolerance response, long-term survival, virulence, and antibiotic resistance. ClpL is poorly characterized and its molecular mechanisms of chaperone activity are largely unclear. Here, we biochemically characterized the ClpL protein from Streptococcus mutans, a dental pathogen, to understand its biological functions. ClpL harbors five domains: N-domain, two nucleotide binding domains (NBD-1 and NBD-2), M-domain, and C-domain. NBD-1 and NBD-2 contain distinct Walker A and B motifs for ATP binding and hydrolysis, respectively. We found that ClpL predominantly exists as a trimer in solution; however, upon ATP binding, it rapidly forms a hexameric structure. To study structure-function activity, we constructed several substitution and deletion mutants. We found that mutations in Walker A and B motifs interfered with the ATP hydrolysis and oligomerization. Similarly, deletions of N-, M-, and C-domains abolished both the ATPase activity and oligomerization. Since we previously found that ClpL acts as a chaperone, we analyzed the chaperone activity. Surprisingly, we found that the NBD-2 mutants did not display any chaperone activity indicating the essentiality of ATP binding and hydrolysis by NBD-2 for chaperone. However, NBD-1 mutants showed chaperone activities, but the activities were variable depending on the nature of the mutations. Our results indicate that unlike other HSP100 family chaperones, ClpL is a novel chaperone that does not require any additional secondary chaperones for its activity.
登录
查看更多内容
影响因子：
5
作者：
Mogk A;Kummer E;Bukau B
通讯作者：
Bukau B
影响因子：
4.8
作者：
Angel Fernandez-Higuero, Jose;Acebron, Sergio P.;Muga, Arturo
通讯作者：
Muga, Arturo
影响因子：
4.2
作者：
Hingston, Patricia;Brenner, Thomas;Wang, Siyun
通讯作者：
Wang, Siyun
影响因子：
3.6
作者：
HUANG, DC;HUANG, XF;NOVEL, M
通讯作者：
NOVEL, M
影响因子：
2.9
作者：
Ihara, Makoto;Matsuura, Noriko;Yamashita, Atsuko
通讯作者：
Yamashita, Atsuko