A New Family of Intrinsically Disordered Proteins: Structural Characterization of the Major Phasin PhaF from Pseudomonas putida KT2440

Abstract

Phasins are intracellular polyhydroxyalkanoat4e (PHA)-associated proteins involved in the stabilization of these bacterial carbon storage granules. Despite its importance in PHA metabolism and regulation, only few reports have focused so far on the structure of these proteins. In this work we have investigated the structure and stability of the PhaF phasin from Pseudomonas putida KT2440, a protein that is involved in PHA granule stabilization and distribution to daughter cells upon cell division. A structural, three-dimensional model of the protein was built from homology modeling procedures and consensus secondary structure predictions. The model predicts that PhaF is an elongated protein, with a long, amphipathic N-terminal helix with PHA binding capacity, followed by a short leucine zipper involved in protein oligomerization and a superhelical C-terminal domain wrapped around the chromosomal DNA. Hydrodynamic, spectroscopical and thermodynamic experiments validated the model and confirmed both that free PhaF is a tetramer in solution and that most part of the protein is intrinsically disordered in the absence of its ligands. The results lay a molecular basis for the explanation of the biological role of PhaF and, along with an exhaustive analysis of phasin sequence databases, suggest that intrinsic disorder and oligomerization through coiled-coils may be a widespread mechanism among these proteins.

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

“…This was found to be a general characteristic of phasins (18)(19)(20). However, the proportion of residues in an ␣-helix conformation was observed to change according to the environment in experiments performed with PhaP Az and the N-terminal domain of PhaF Pp , as this proportion was shown to increase in the presence of structurestabilizing solvents, like 2,2,2-trifluoroethanol, or with the addition of sodium oleate, which creates a hydrophobic environment that mimics PHB (18,19).…”

“…Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins, such as PhaP from R. eutropha (PhaP Re ) (17), PhaP from Azotobacter sp. strain FA8 (PhaP Az ) (18), PhaF from P. putida (PhaF Pp ) (19), PhaP from Aeromonas hydrophila (PhaP Ah ) (20), and PhaP from Synechocystis sp. PCC 6803 (PhaP Sp ) (12).…”

“…A structural feature that has been recently recognized in phasins is the presence of disordered regions, which are predicted in many of these proteins (18,19). Intrinsically disordered proteins or disordered regions are interconverting dynamic ensembles of structures that do not fold in a single structure and are important for the regulatory functions of the proteins because of their highly flexible nature (21).…”

“…Intrinsically disordered proteins or disordered regions are interconverting dynamic ensembles of structures that do not fold in a single structure and are important for the regulatory functions of the proteins because of their highly flexible nature (21). Maestro et al proposed that the multifunctional phasin PhaF Pp belonged to a new family of intrinsically disordered proteins due to the unstructured regions predicted in its N-terminal domain, which was also predicted to contain a long ␣-helix that is partially disordered in the absence of PHA (19). Unstructured regions were also found in PhaP Az .…”

“…All phasins form oligomers, and most studies have indicated that these proteins are normally tetramers in solution (12,(18)(19)(20). However, Neumann et al proposed that PhaP Re occurs as a trimer (17), and some phasins have been reported to form other kinds of oligomers, such as dimers and dodecamers (corresponding to PhaP5 and PhaM from R. eutropha, respectively) (22).…”