Structural bioinformatics of bacterial outer membrane beta-barrel enzymes and their AlphaFold2 predicted water-soluble QTY variants

Beta-barrel enzymes are an important area of study in the field of structural biology. These proteins serve crucial roles, acting as porins, transporters, enzymes, virulence factors, and receptors. Recent research has unveiled a novel role for beta-barrel enzymes in the bacterial outer membrane as sentinels. They remain inactive when the outer membrane is intact but activate in response to host immune responses and antibiotics that breach this barrier. Understanding their structure and function is pivotal in grasping their sentinel role in the bacterial outer membrane. Here we present our structural bioinformatics analyses on four bacterial outer membrane beta-barrel enzymes: a) OMPLA, b) OmpT, c) PagP from E.coli, and d) PagL from Pseudomonas aeruginosa. We previously applied the QTY code to convert hydrophobic alpha-helices and beta-barrels into hydrophilic forms in over 50 membrane proteins. The QTY code directly swaps hydrophobic amino acids leucine (L), isoleucine (I), valine (V), and phenylalanine (F) with hydrophilic glutamine (Q), threonine (T), and tyrosine (Y). We superposed the structures of native beta-barrel outer membrane enzymes with their AlphaFold2-predicted QTY variant structures that showed remarkable similarity despite the replacement of at least 22.95% amino acids in transmembrane regions, the superposed structures displayed notable structural similarity, indicated by RMSD values ranging from 0.181Å to 0.286Å. We also analyze the hydrophobicity patches and the enhanced hydrophilic surfaces. Our research provide insights into the structural similarity of hydrophobic and hydrophilic beta-barrel enzymes, validating the utility of the QTY code for investigating beta-barrel membrane enzymes. Our results not only demonstrate that the QTY code serves as a straightforward tool for designing water-soluble membrane proteins across various biological contexts, but it may also stimulate experiments to validate our structural bioinformatic studies.

Structural bioinformatics of bacterial integral membrane enzymes and their AlphaFold2 predicted water-soluble QTY variants

Integral Membrane enzymes (IMPs) are proteins that are essential for cells to function properly. They are embedded in the membrane that surrounds cells and allow important processes to happen [48]. However, studying these proteins has been difficult because they don't dissolve well in water and tend to aggregate together when removed from the membrane. In this work, we look at seven membrane enzyme proteins that are "alpha helix" shape. We use highly accurate computer predictions of their 3D structures from a program called AlphaFold2. Previously, we developed a code called QTY that makes alpha-helix, barrel, and antibody proteins more water-soluble by replacing out some water-insoluble amino acids (leucine, isoleucine, valine and phenylalanine) for water-soluble ones like (glutamine, threonine, and tyrosine). We found that even with over 41% of the amino acids swapped using the QTY code, the predicted 3D structures of the water-soluble variants are extremely similar to the original membrane versions, with very small deviations. We also analyse how the surfaces of these proteins become more water-friendly and where patches of water-insoluble areas remain. Our study shows that water-soluble QTY variants and membrane-bound alpha-helical enzyme proteins have comparable structures, proving the QTY code works for making water-soluble variants of these proteins. Not only does this provide a simple way to study membrane proteins in water, but it encourages further experiments to test our computer predictions in the lab.