Structural bioinformatics of three Epstein-Barr Virus (EBV) Integral Membrane Proteins and their water-soluble QTY analogs

The Epstein-Barr virus (EBV) is a highly prevalent virus worldwide that is associated with several lymphoid and epithelial malignancies. However, extensive research on EBV integral membrane proteins BILF1, LMP1 and LMP2, has been scarce due to their hydrophobic transmembrane domains. Our study applies the QTY code (glutamine, threonine, tyrosine) to design water-soluble analogs of BILF1, LMP1 and LMP2 with reduced hydrophobicity, where we systematically replaced hydrophobic amino acid residues leucine (L), isoleucine (I), valine (V), and phenylalanine (F) with structurally similar polar residues glutamine (Q), threonine (T), and tyrosine (Y). We retrieved their native sequences from UniProt, identified transmembrane domains using Protter, then performed QTY design through the Protein Solubilizing Server (PSS). We then predicted native and QTY structures using in silico prediction tools AlphaFold3, ColabFold, and Boltz-2. Our analyses demonstrate that despite significant protein sequence replacements in their transmembrane domains (54.15%-61.59%) and increased intrinsic solubility, the QTY analogs exhibited minimal changes in isoelectric point (0.00-0.15 decrease) and molecular weight (0.7-1.2 kDa increase). Additionally, structural superpositions between QTY analogs and native structures using PyMOL yield low RMSD values (0.217[A] -1.202[A]). Our results demonstrate the QTY codes ability to design detergent-free analogs of BILF1, LMP1 and LMP2 with substantially reduced hydrophobicity and aggregation propensity whilst preserving native-like structures. Our results may facilitate protein characterization studies, therapeutic research on EBV, and other protocols that typically require protein solubilization.