title: Promoting protein self-association in non-glycosylated Thermomyces lanuginosus lipase based on crystal lattice contacts.
abstract: 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 (>2M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.
authors.affiliation: iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark; Zealand Pharma A/S, Smedeland 36, DK-2600 Glostrup, Denmark.
authors.forename: Jens Kvist
authors.initials: JK
authors.lastname: Madsen
authors.affiliation: iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark; Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.
authors.forename: Thomas Rebsdorf
authors.initials: TR
authors.lastname: Sørensen
authors.affiliation: iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark; Department of Chemistry, Aarhus University, Langelandsgade 140, DK-8000 Aarhus C, Denmark.
authors.forename: Jørn Døvling
authors.initials: JD
authors.lastname: Kaspersen
authors.affiliation: Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.
authors.forename: Maria Berggård
authors.initials: MB
authors.lastname: Silow
authors.affiliation: Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Vind
authors.affiliation: iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark; Department of Chemistry, Aarhus University, Langelandsgade 140, DK-8000 Aarhus C, Denmark.
authors.forename: Jan Skov
authors.initials: JS
authors.lastname: Pedersen
authors.affiliation: Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark. Electronic address: asv@novozymes.com.
authors.forename: Allan
authors.initials: A
authors.lastname: Svendsen
authors.affiliation: iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark. Electronic address: dao@inano.au.dk.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Otzen
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D002850:Chromatography, Gel; D006031:Glycosylation; D008049:Lipase / Q000378:metabolism*; D012542:Scattering, Radiation
chemical_list: D008049:Lipase
keywords: Enzyme activity; Gel filtration; Lattice contacts; Lipase; Oligomers; Protein design; Saxs