Insights into Biofilm Dispersal Regulation from the Crystal Structure of the PAS-GGDEF-EAL Region of RbdA from Pseudomonas aeruginosa
Insights into Biofilm Dispersal Regulation from the Crystal Structure of the PAS-GGDEF-EAL Region of RbdA from Pseudomonas aeruginosa
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DOI：
10.1128/jb.00515-17
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发表时间：
2018-02-01
影响因子：
3.2
通讯作者：
Lescar, Julien
中科院分区：
文献类型：
--
作者：
Liu, Chong;Liew, Chong Wai;Lescar, Julien
RbdA 是铜绿假单胞菌生物膜分散的正调节剂。其胞质区 (cRbdA) 包含 N 端 Per-ARNT-Sim (PAS) 结构域，随后是二鸟苷酸环化酶 (GGDEF) 结构域和 EAL 结构域，其磷酸二酯酶活性通过 GTP 与 GGDEF 结构域的结合而变构刺激。我们报告了 cRbdA 和两种二元复合物的晶体结构：一种具有与 GGDEF 活性位点结合的 GTP/Mg2+，另一种具有与 c-di-GMP 底物结合的 EAL 结构域。这些结构揭示了由紧密堆积的 N 端 PAS 结构域和非规范 EAL 二聚体稳定的 2 重对称二聚体。自抑制开关由紧邻 GGDEF 结构域 N 端的 α 螺旋（S 螺旋）形成，该螺旋与其他 EAL 单体的 EAL 二聚化螺旋 (α(6-E)) 相互作用，并将蛋白质维持在锁定构象。我们提出，如小角 X 射线散射 (SAXS) 实验所示，GTP 结合后 cRbdA 的局部构象变化导致 PAS 结构域和 S 螺旋偏离 GGDEF-EAL 结构域的结构。连接 GGDEF 和 EAL 区域的 α 螺旋杆（H 螺旋）的存在应促进结构域重新定向，从而使 EAL 结构域重新排列成活性二聚体构象。 重要性 细菌病原体形成的生物膜会增加对抗生素的耐药性。 RbdA 正向调节铜绿假单胞菌的生物膜分散。这里展示的 RbdA 蛋白细胞质区域的晶体结构揭示了两个进化上保守的螺旋在调节 RbdA 活性中发挥着重要作用，这对在几种细菌病原体的蛋白质组中丰富的其他 GGDEF-EAL 双结构域具有影响。因此，这项工作可能有助于开发促进细菌生物膜分散的小分子。
RbdA is a positive regulator of biofilm dispersal of Pseudomonas aeruginosa. Its cytoplasmic region (cRbdA) comprises an N-terminal Per-ARNT-Sim (PAS) domain followed by a diguanylate cyclase (GGDEF) domain and an EAL domain, whose phosphodiesterase activity is allosterically stimulated by GTP binding to the GGDEF domain. We report crystal structures of cRbdA and of two binary complexes: one with GTP/Mg2+ bound to the GGDEF active site and one with the EAL domain bound to the c-di-GMP substrate. These structures unveil a 2-fold symmetric dimer stabilized by a closely packed N-terminal PAS domain and a noncanonical EAL dimer. The autoinhibitory switch is formed by an alpha-helix (S-helix) immediately N-terminal to the GGDEF domain that interacts with the EAL dimerization helix (alpha(6-E)) of the other EAL monomer and maintains the protein in a locked conformation. We propose that local conformational changes in cRbdA upon GTP binding lead to a structure with the PAS domain and S-helix shifted away from the GGDEF-EAL domains, as suggested by small-angle X-ray scattering (SAXS) experiments. Domain reorientation should be facilitated by the presence of an alpha-helical lever (H-helix) that tethers the GGDEF and EAL regions, allowing the EAL domain to rearrange into an active dimeric conformation.IMPORTANCE Biofilm formation by bacterial pathogens increases resistance to antibiotics. RbdA positively regulates biofilm dispersal of Pseudomonas aeruginosa. The crystal structures of the cytoplasmic region of the RbdA protein presented here reveal that two evolutionarily conserved helices play an important role in regulating the activity of RbdA, with implications for other GGDEF-EAL dual domains that are abundant in the proteomes of several bacterial pathogens. Thus, this work may assist in the development of small molecules that promote bacterial biofilm dispersal.