Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas, CSIC, Madrid 28040, Spain.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche 03202, Spain.
Biochim Biophys Acta Gen Subj. 2019 Feb;1863(2):362-370. doi: 10.1016/j.bbagen.2018.11.002. Epub 2018 Nov 9.
Phasins are low molecular mass proteins that accumulate strongly in bacterial cells in response to the intracellular storage of polyhydroxyalkanoates (PHA). Although lacking catalytic activity, phasins are the major components of the surface of the PHA granules and could be potentially involved in the formation of a network-like protein layer surrounding the polyester inclusions. Structural models revealed phasins to possess coiled-coil regions that might be important in the establishment of protein-protein interactions. However, there is not experimental evidence of a coiled-coil mediated oligomerization in these proteins.
Structure prediction analyses were used to characterize the coiled-coil motifs of phasins PhaF and PhaI -produced by the model bacterium Pseudomonas putida KT2440-. Their oligomerization was evaluated by biolayer interferometry and the in vivo two-hybrid (BACTH) system. The interaction ability of a series of coiled-coil mutated derivatives was also measured.
The formation of PhaF and PhaI complexes was detected. A predicted short leucine zipper-like coiled-coil (ZIP), containing "ideal" residues located within the hydrophobic core, was shown responsible for the oligomers stability. The substitution of key residues (leucines or valines) in PhaI ZIP (ZIPI) for alanine reduced by four fold the oligomerization efficiency.
These results indicate that coiled-coil motifs are essential for phasin interactions. Correct oligomerization requires the formation of a stable hydrophobic interface between both phasins.
Our findings elucidate the oligomerization motif of PhaF and PhaI. This motif is present in most phasins from PHA-accumulating bacteria and offers a potentially important target for modulating the PHA granules stability.
类phasins 是低分子量的蛋白质，在细胞内聚积多羟基烷酸（PHA）时会强烈积累。虽然缺乏催化活性，但类phasins 是 PHA 颗粒表面的主要成分，可能参与聚酯包裹体周围的网络状蛋白层的形成。结构模型表明类phasins 具有卷曲螺旋区域，这可能对建立蛋白-蛋白相互作用很重要。然而，目前还没有这些蛋白中卷曲螺旋介导的寡聚化的实验证据。
使用结构预测分析来描述模型菌假单胞菌 KT2440 产生的类phasins PhaF 和 PhaI 的卷曲螺旋基序。通过生物层干涉测量法和体内双杂交（BACTH）系统评估它们的寡聚化。还测量了一系列卷曲螺旋突变衍生物的相互作用能力。
检测到 PhaF 和 PhaI 复合物的形成。预测的短亮氨酸拉链样卷曲螺旋（ZIP），包含位于疏水区核心内的“理想”残基，负责寡聚体的稳定性。PhaI ZIP（ZIPI）中关键残基（亮氨酸或缬氨酸）替换为丙氨酸会使寡聚化效率降低四倍。
这些结果表明卷曲螺旋基序对于phasins 相互作用至关重要。正确的寡聚化需要两个 phasins 之间形成稳定的疏水面。
我们的发现阐明了 PhaF 和 PhaI 的寡聚化基序。该基序存在于大多数聚羟基脂肪酸（PHA）积累细菌的类phasins 中，为调节 PHA 颗粒稳定性提供了一个潜在的重要目标。
