We have used the crystal structure of Thermomyces lanuginosus lipase (TlL) to identify and strengthen potential protein–protein interaction sites in solution. As wildtype we used a deglycosylated mutant of TlL (N33Q). We designed a number of TlL mutants to promote interactions via interfaces detected in the crystal-lattice structure, through strengthening of hydrophobic, polar or electrostatic contacts or truncation of sterically blocking residues. We identify a mutant predicted to lead to increased interfacial hydrophobic contacts (N92F) that shows markedly increased self-association properties on native gradient gels. While wildtype TlL mainly forms monomer and < 5% dimers, N92F forms stable trimers and dimers according to Size-Exclusion Chromatography and Small-Angle X-ray Scattering. These oligomers account for ~ 25% of the population and their enzymatic activity is comparable to that of the monomer. Self-association stabilizes TlL against thermal denaturation. Furthermore, the trimer is stable to dilution and requires high concentrations (> 2 M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.
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