Allosteric communication network in the tetrameric restriction endonuclease Bse634I.
Restriction endonuclease Bse634I is a homotetramer arranged as a dimer of two primary dimers. Bse634I displays its maximum catalytic efficiency upon binding of two copies of cognate DNA, one per each primary dimer. The catalytic activity of Bse634I on a single DNA copy is down-regulated due to the cross-talking interactions between the primary dimers. The mechanism of signal propagation between the individual active sites of Bse634I remains unclear. To identify communication pathways involved in the catalytic activity regulation of Bse634I tetramer we mutated a selected set of amino acid residues at the dimer-dimer interface and analysed the oligomeric state and catalytic properties of the mutant proteins. We demonstrate that alanine replacement of N262 and V263 residues located in the loop at the tetramerisation interface did not inhibit tetramer assembly but dramatically altered the catalytic properties of Bse634I despite of the distal location from the active site. Kinetic analysis using cognate hairpin oligonucleotide and one and two-site plasmids as substrates allowed us to identify two types of communication signals propagated through the dimer-dimer interface in the Bse634I tetramer: the inhibitory, or "stopper" and the activating, or "sync" signal. We suggest that the interplay between the two signals determines the catalytic and regulatory properties of the Bse634I and mutant proteins.
Zaremba
Mindaugas
M
Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania.
Sasnauskas
Giedrius
G
Urbanke
Claus
C
Siksnys
Virginijus
V
D000494:Allosteric Regulation / Q000187:drug effects; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010957:Plasmids / Q000737:chemistry; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
D004247:DNA; D004262:DNA Restriction Enzymes; D019791:Guanidine