Characterization and Diversification of AraC/XylS Family Regulators Guided by Transposon Sequencing

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids

Foldy:An open-source web application for interactive protein structure analysis

Foldy is a cloud-based application that allows non-computational biologists to easily utilize advanced AI-based structural biology tools, including AlphaFold and DiffDock. With many deployment options, it can be employed by individuals, labs, universities, and companies in the cloud without requiring hardware resources, but it can also be configured to utilize locally available computers. Foldy enables scientists to predict the structure of proteins and complexes up to 6000 amino acids with AlphaFold, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. In our manuscript, we detail Foldy’s interface design, deployment strategies, and optimization for various user scenarios. We demonstrate its application through case studies including rational enzyme design and analyzing proteins with domains of unknown function. Furthermore, we compare Foldy’s interface and management capabilities with other open and closed source tools in the field, illustrating its practicality in managing complex data and computation tasks. Our manuscript underlines the benefits of Foldy as a day-to-day tool for life science researchers, and shows how Foldy can make modern tools more accessible and efficient.</p

Foldy: An open-source web application for interactive protein structure analysis.

Foldy is a cloud-based application that allows non-computational biologists to easily utilize advanced AI-based structural biology tools, including AlphaFold and DiffDock. With many deployment options, it can be employed by individuals, labs, universities, and companies in the cloud without requiring hardware resources, but it can also be configured to utilize locally available computers. Foldy enables scientists to predict the structure of proteins and complexes up to 6000 amino acids with AlphaFold, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. In our manuscript, we detail Foldy's interface design, deployment strategies, and optimization for various user scenarios. We demonstrate its application through case studies including rational enzyme design and analyzing proteins with domains of unknown function. Furthermore, we compare Foldy's interface and management capabilities with other open and closed source tools in the field, illustrating its practicality in managing complex data and computation tasks. Our manuscript underlines the benefits of Foldy as a day-to-day tool for life science researchers, and shows how Foldy can make modern tools more accessible and efficient

REC protein family expansion by the emergence of a new signaling pathway

This report presents multi-genome evidence that REC protein family expansion occurs when the emergence of new pathways gives rise to functional discordance. Specificity between residues in REC domain containing response regulators with paired histidine kinases is under negative purifying selection, constrained by the presence of other bacterial two-component systems signaling cascades that share sequence and structural identity. Presuming that the two-component systems can evolve by neutral amino acid changes (neutral drift) when purifying evolutionary constraints are relaxed, how might the REC protein family expand by amino acid changes when these constraints remain intact? Using an unsupervised machine learning approach to observe the sequence landscape of REC domains across long phylogenetic distances, we find that within-gene recombination, a subcategory of gene conversion, switched the effector domain and, consequently, the regulatory context of a duplicated response regulator from transcriptional regulation by σ54 to that by σ70. We determined that the recombined response regulator diverged from its parent by episodic diversifying selection and neutral drift. Functional experiments of the parent of recombined response regulators in a model Pseudomonas putida KT2440 model system revealed that the parent and recombined response regulators sense and respond to different carboxylic acids. Finally, a residue-switching experiment using structural predictions and functional characterization suggests that the new residues in the recombined regulator could form a new interaction interface and mediate condition-specific phosphotransfer. Overall, our study finds that genetic perturbations can create conditions of functional discordance, whereby the REC protein family can evolve by episodic diversifying selection

Comparison of different structure prediction tools.

Comparison of different structure prediction tools.</p

A fatty acyl-AMP ligase with substrate docked.

The structure of the wild type FadD23 from M. smegmatis as predicted by AlphaFold in Foldy is shown in blue, rendered in pymol. Within the active site is the pose of octanoyl-AMP docked as predicted by AutoDock Vina in Foldy. This pose was chosen from the ensemble of predicted poses based on biochemical information about the active site. Two notable residues are highlighted: G321 and L218. The wild type backbone and side chain are displayed for both in blue. Displayed in orange and red are the same residues taken from two different structures which are mutants of the wild type. On top is residue 321 from the G321W mutant’s structure as predicted by AlphaFold. This mutant seems to occlude the proper insertion of the C8 ligand. Below is residue 218 from the L218W mutant’s structure as predicted by AlphaFold. This mutation seems to allow insertion of the C8 ligand but would occlude insertion of longer ligands like the C10-AMP. Based on these three structures, L218W is a good candidate for making an FAAL which prefers shorter chain fatty acyl AMPs while preserving activity for the C8 chains.</p

Structure View.

The Structure view is a window into a predicted structure and a host of tools and associated information.</p

Putative complex formation of two domains of unknown function in <i>P</i>. <i>putida</i>.

Four genes known to be essential for metabolism of Tween-20 were co-folded with AlphaFold in Foldy. A. Peptide-Peptide Contact Probability: As done in Humphreys et al [6] Foldy approximates the peptide-peptide contact probability as the maximum contact probability of any residues between two cofolded peptides. The matrix of peptide-peptide contact probabilities is shown in the Contact tab of the Structure View page. This contact probability matrix shows that maybe: PP_0766 and PP_2019 weakly interact, PP_0766 and PP_0765 weakly interact, and PP_2019 and PP_2018 strongly interact. B. Predicted Interactions of Phenotypically Related Proteins: The four proteins whose knockouts have related phenotypes were folded in Foldy, and although all four cluster closely, the peptide-peptide contact probability map indicates that not all four interact directly with each other. By using biochemical knowledge about these proteins, we are led to suspect that there are two different complexes which can form. C. Novel Hypothesized Substrate Transport System: Two DUFs in P. putida, previously hypothesized to have hydrolase activity may actually be involved in substrate transport: PP_0765 (DUF1302, top blue) and PP_0766 (DUF1329, top green, bottom green). PP_0765 has the characteristic beta-barrel of a membrane protein, and shows high likelihood of forming a complex with PP_0766 (top). Additionally, PP_0766 is predicted to form a heterotrimer with PP_2018 (bottom blue) and PP_2019 (bottom red).</p