Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses.

•Novel use of yeast-display for MD-2 expression enabled rapid mutational analyses.•Selection of MD-2 mutants from libraries revealed importance of specific residues.•Aromatic residues were strongly preferred at F119 and F121 for LPS binding.•Proline mutations selected in a key loop (119-132) stabilized the protein.•Charged mutations at S120 and G123 yielded enhanced ligand binding.