DNA facilitates heterodimerization between human transcription factors FoxP1 and FoxP2 by increasing their conformational flexibility.

Transcription factors regulate gene expression by binding to DNA. They have disordered regions and specific DNA-binding domains. Binding to DNA causes structural changes, including folding and interactions with other molecules. The FoxP subfamily of transcription factors in humans is unique because they can form heterotypic interactions without DNA. However, it is unclear how they form heterodimers and how DNA binding affects their function. We used computational and experimental methods to study the structural changes in FoxP1's DNA-binding domain when it forms a heterodimer with FoxP2. We found that FoxP1 has complex and diverse conformational dynamics, transitioning between compact and extended states. Surprisingly, DNA binding increases the flexibility of FoxP1, contrary to the typical folding-upon-binding mechanism. In addition, we observed a 3-fold increase in the rate of heterodimerization after FoxP1 binds to DNA. These findings emphasize the importance of structural flexibility in promoting heterodimerization to form transcriptional complexes.

Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate.

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.

Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures.

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60°C and 70°C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25°C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases. IMPORTANCE A myriad of consumer products contains polyethylene terephthalate (PET), a plastic that has accumulated as waste in the environment due to its long-term stability and poor waste management. One promising solution is the enzymatic biodegradation of PET, with most known enzymes only catalyzing this process at high temperatures. Here, we bioinformatically identified and biochemically characterized an enzyme from an Antarctic organism that degrades PET at 25°C with similar efficiency to the few PET-degrading enzymes active at moderate temperatures. Reasoning that Antarctica harbors other PET-degrading enzymes, we analyzed available data from Antarctic metagenomic samples and successfully identified other potential enzymes. Our findings contribute to increasing the repertoire of known PET-degrading enzymes that are currently being considered as biocatalysts for the biological recycling of plastic waste.

Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.