Role of leucine zipper-like motifs in the oligomerization of Pseudomonas putida phasins

Abstract Background 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. Methods 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. Results 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. Conclusions 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. General Significance. 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.