Structure-function dissection of Myxococcus xanthus CarD N-terminal domain, a defining member of the CarD_CdnL_TRCF family of RNA polymerase interacting proteins.
Structure-function dissection of Myxococcus xanthus CarD N-terminal domain, a defining member of the CarD_CdnL_TRCF family of RNA polymerase interacting proteins.
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DOI：
10.1371/journal.pone.0121322
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Elías-Arnanz M
中科院分区：
文献类型：
--
作者：
Bernal-Bernal D;Gallego-García A;García-Martínez G;García-Heras F;Jiménez MA;Padmanabhan S;Elías-Arnanz M
细菌RNA聚合酶(RNAP)结合蛋白CARD_CDNL_TRCF家族的两个原型，粘菌黄色葡萄球菌CARD和CDNL，具有不同的功能，其分子基础仍不清楚。CARD是一种与多种胞外功能σ因子的作用相关联的全球调节因子，它通过N-末端结构域CarDNt与β亚单位(β-RNAP-DNAg)结合，并通过与真核高迁移率A组(HMGA)蛋白相似的C-末端结构域与DNAs结合。CDn1是一种与CarDNt类似的蛋白，对细胞活力至关重要，它参与了RNAPA依赖的σ启动子的激活，并与β相互作用，但不与Carg相互作用。虽然CarDNt本身的HMGA样结构域本身是不活跃的，但我们发现CarDNt在体内激活ECFDNA依赖的启动子的能力很低，这表明C端的σ结合域是最大化活性所必需的。我们对CarDNt的结构和功能的剖析揭示了一个N末端的五链β-Sheet都铎结构域CarD_1-72，其结构和与RNAP-β的接触都与CDN_1相似。有趣的是，与CDNL形成鲜明对比的是，破坏其与RNAP-β相互作用的CARD突变并没有使活性失效。我们的数据表明，CarDNt的C-末端片段CarD61-179可能在结构上与CDNL的对应物不同，它至少包含两个不同的关键功能决定因素：(A)Carg结合，它是CARD所特有的；(B)基本残基拉伸，这在CDNL中也是保守的和功能所需的。这项研究强调了相似蛋白质模块中共享和发散相互作用的演变，这些相互作用使一个功能重要和广泛存在的细菌蛋白质家族中两个相关成员的不同活动得以实现。
Two prototypes of the large CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-binding proteins, Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP β subunit (RNAP-β) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-β but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded β -sheet Tudor-like domain, CarD1–72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61–179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family.
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影响因子：
3.6
作者：
Garner AL;Weiss LA;Manzano AR;Galburt EA;Stallings CL
通讯作者：
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影响因子：
3.7
作者：
Gallego-García A;Mirassou Y;García-Moreno D;Elías-Arnanz M;Jiménez MA;Padmanabhan S
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影响因子：
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