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.

RfaH, a two-domain protein from a universally conserved NusG/Spt5 family of regulators, is required for the transcription and translation of long virulence and conjugation operons in many Gram-negative bacterial pathogens. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA element. Upon recruitment, the C-terminal domain of RfaH refolds from an a-hairpin, which is bound to RNA polymerase binding site within the N-terminal domain, into an unbound b-barrel that interacts with the ribosome. Although structures of the autoinhibited (a-hairpin) and active (b-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state ($20% deuteron incorporation), whereas the rest of this domain is highly flexible (>40% deuteron incorporation), and its flexibility only decreases in the b-folded state. Computationally predicted DG agree with these results by displaying similar anisotropic stability within the tip of the a-hairpin and on neighboring N-terminal domain residues. Remarkably, the b-folded state shows comparable structural flexibility than nonmetamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins and for devising targeted strategies to combat bacterial infections.

The COVID-19 pandemic has swiftly forced a change in learning strategies across educational institutions, from extensively relying on in-person activities toward online teaching. It is particularly difficult to adapt courses that depend on physical equipment to be now carried out remotely. This is the case for bioinformatics, which typically requires dedicated computer classrooms, as the logistics of granting remote access to a workstation or relying on the computational resources of each student is not trivial. A possible workaround is using cloud server-based computing resources, such as Google Colaboratory, a free web browser application that allows the writing and execution of Python programming through Jupyter notebooks, integrating text, images, and code cells. Following a cloud-based approach, we migrated the practical activities of a course on molecular modeling and simulation into the Google Colaboratory environment resulting in 12 tutorials that introduce students to topics such as phylogenetic analysis, molecular modeling, molecular docking, several flavors of molecular dynamics, and coevolutionary analysis. Each of these notebooks includes a brief introduction to the topic, software installation, execution of the required tools, and analysis of results, with each step properly described. Using a Likert scale questionnaire, a pool of students positively evaluated these tutorials in terms of the time required for their completion, their ability to understand the content and exercises developed in each session, and the practical significance and impact that these computational tools have on scientific research. All tutorials are freely available at https://github.com/pb3lab/ibm3202.