Mutagenesis and homology modeling of the Tn21 integron integrase IntI1.
Horizontal DNA transfer between bacteria is widespread and a major cause of antibiotic resistance. For logistic reasons, single or combined genes are shuttled between vectors such as plasmids and bacterial chromosomes. Special elements termed integrons operate in such shuttling and are therefore vital for horizontal gene transfer. Shorter elements carrying genes, cassettes, are integrated in the integrons, or excised from them, by virtue of a recombination site, attC, positioned in the 3' end of each unit. It is a remarkable and possibly restricting elementary feature of attC that it must be single-stranded while the partner target site, attI, may be double-stranded. The integron integrases belong to the tyrosine recombinase family, and this work reports mutations of the integrase IntI1 from transposon Tn21, chosen within a well-conserved region characteristic of the integron integrases. The mutated proteins were tested for binding to a bottom strand of an attC substrate, by using an electrophoresis mobility shift assay. To aid in interpreting the results, a homology model was constructed on the basis of the crystal structure of integron integrase VchIntIA from Vibrio cholerae bound to its cognate attC substrate VCRbs. The local stability and hydrogen bonding network of key domains of the modeled structure were further examined using molecular dynamics simulations. The homology model allowed us to interpret the roles of several amino acid residues, four of which were clearly binding assay responsive upon mutagenesis. Notably, we also observed features indicating that IntI1 may be more prone to base-specific contacts with VCRbs than VchIntIA.
Johansson
Carolina
C
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 582, 751 23 Uppsala, Sweden.
Boukharta
Lars
L
Eriksson
Jens
J
Aqvist
Johan
J
Sundström
Lars
L
D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001345:Autoradiography; D055162:Biocatalysis; D004247:DNA / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006860:Hydrogen Bonding; D019426:Integrases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011995:Recombination, Genetic; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D014734:Vibrio cholerae / Q000201:enzymology*
D004251:DNA Transposable Elements; D050505:Mutant Proteins; D004247:DNA; D019426:Integrases; C115006:integron integrase IntI1