Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, 13083-970, Campinas, SP, Brazil.
Proteins. 2010 Dec;78(16):3386-95. doi: 10.1002/prot.22846. Epub 2010 Sep 16.
Many plant pathogenic bacteria rely on effector proteins to suppress defense and manipulate host cell mechanisms to cause disease. The effector protein PthA modulates the host transcriptome to promote citrus canker. PthA possesses unusual protein architecture with an internal region encompassing variable numbers of near-identical tandem repeats of 34 amino acids termed the repeat domain. This domain mediates protein-protein and protein-DNA interactions, and two polymorphic residues in each repeat unit determine DNA specificity. To gain insights into how the repeat domain promotes protein-protein and protein-DNA contacts, we have solved the structure of a peptide corresponding to 1.5 units of the PthA repeat domain by nuclear magnetic resonance (NMR) and carried out small-angle X-ray scattering (SAXS) and spectroscopic studies on the entire 15.5-repeat domain of PthA2 (RD2). Consistent with secondary structure predictions and circular dichroism data, the NMR structure of the 1.5-repeat peptide reveals three α-helices connected by two turns that fold into a tetratricopeptide repeat (TPR)-like domain. The NMR structure corroborates the theoretical TPR superhelix predicted for RD2, which is also in agreement with the elongated shape of RD2 determined by SAXS. Furthermore, RD2 undergoes conformational changes in a pH-dependent manner and upon DNA interaction, and shows sequence similarities to pentatricopeptide repeat (PPR), a nucleic acid-binding motif structurally related to TPR. The results point to a model in which the RD2 structure changes its compactness as it embraces the DNA with the polymorphic diresidues facing the interior of the superhelix oriented toward the nucleotide bases.
许多植物病原细菌依赖效应蛋白来抑制防御并操纵宿主细胞机制以引起疾病。效应蛋白 PthA 调节宿主转录组以促进柑橘溃疡病。PthA 具有不寻常的蛋白质结构，其内部区域包含数量不定的近相同串联重复 34 个氨基酸的重复域。该结构域介导蛋白质-蛋白质和蛋白质-DNA 相互作用，每个重复单元中的两个多态性残基决定 DNA 特异性。为了深入了解重复域如何促进蛋白质-蛋白质和蛋白质-DNA 接触，我们通过核磁共振（NMR）解决了对应于 PthA 重复域 1.5 个单位的肽的结构，并对 PthA2（RD2）的整个 15.5 个重复域进行了小角度 X 射线散射（SAXS）和光谱研究。与二级结构预测和圆二色性数据一致，1.5 个重复肽的 NMR 结构揭示了三个由两个转角连接的α-螺旋，折叠成四肽重复（TPR）样结构域。NMR 结构证实了 RD2 理论上的 TPR 超螺旋，这也与 SAXS 确定的 RD2 的伸长形状一致。此外，RD2 以 pH 依赖性方式和在与 DNA 相互作用时发生构象变化，并显示与五肽重复（PPR）的序列相似性，PPR 是一种与 TPR 结构相关的核酸结合基序。结果表明，当 RD2 结构与 DNA 结合时，其紧凑性会发生变化，具有多态二残基的超螺旋内部面向核苷酸碱基。
