title: Structural Determinants of Sleeping Beauty Transposase Activity.
abstract: Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.|Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Abrusán
authors.affiliation: Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California, USA.|Present address: Gilead Sciences Inc., Foster City, California, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Yant
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Szilágyi
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Marsh
authors.affiliation: Instistute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Lajos
authors.initials: L
authors.lastname: Mátés
authors.affiliation: Max Delbrück Center for Molecular Medicine, Berlin, Germany.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvák
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Barabás
authors.affiliation: Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D017124:Conserved Sequence; D004251:DNA Transposable Elements; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D055550:Protein Stability; D013329:Structure-Activity Relationship; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D004251:DNA Transposable Elements; D019895:Transposases