Global conformational changes induced by the removal of the carboxyl group of D456 in the cleavage scaffold of nickase B
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CTURE OF MACROMOLECULAR COMPOUNDS
Global Conformational Changes Induced by the Removal of the Carboxyl Group of D456 in the Cleavage Scaffold of Nickase BspD6I: Structural and Electrostatic Analysis G. S. Kachalovaa,b,*, A. N. Popovc, A. K. Yunusovad, R. I. Artyukhd,**, T. A. Perevyazovad, L. A. Zheleznayad, and B. P. Atanasove a
National Research Centre “Kurchatov Institute,” Moscow, 123098 Russia Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences, Moscow, 119071 Russia c European Synchrotron Radiation Facility, Grenoble, 38000 France d Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia eInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, 1113 Bulgaria *e-mail: [email protected] **e-mail: [email protected] b
Received May 29, 2017
Abstract—The three-dimensional structure of the D456A mutant of the nicking endonuclease Nt.BspD6I was determined. According to the concept of the cleavage scaffold, the replacement of D456 by A456, which resulted in complete (100%) loss of nickase activity, was shown to be a trigger of structural changes in the cleavage-scaffold region. Besides, the displacement of Е482 and the rotation of Н449 toward the N-terminal domain initiate conformational changes in the D1 recognition subdomain of the N-terminal domain with the result that the centers of mass of the С- and N-terminal domains are brought into close proximity to each other. Electrostatic calculations showed that changes in the free energy and electrostatic interactions for the mutant nickase are distributed predominantly in the N-terminal domain and that these changes are not attenuated in a radial fashion away from the mutation site but have a distinct direction. DOI: 10.1134/S1063774517060141
INTRODUCTION Nicking endonucleases (nickases) belong to a relatively new class of enzymes. Like restriction endonucleases, these enzymes recognize a short nucleotide sequence (site) in double-stranded DNA with high specificity. However, as opposed to restriction endonucleases, which make double-strand scissions in DNA, nickases hydrolyze only one, predetermined, DNA strand at a fixed (for each nickase) position relative to the recognition site. According to the accepted nomenclature [1], nickases are designated by the symbol N before the abbreviated name of the strain. Depending on which (top or bottom) strand the nickase cleaves, it is designated as Nt or Nb, respectively. Initially, nicking endonucleases were discovered as separate enzymes [2, 3]. The nickase Nt.BspD6I was found in the Bacillus species D6 strain [4]. It recognizes the 5'–GAGTC–3'/5'–GACTC–3' site in double-stranded DNA and cleaves only the DNA strand containing the GAGTC sequence four nucleotide base pairs away on the 3' side of the recognition site. Earlier, site-specific nickases were suggested to be natu-
rally mutated restriction endonucleases, which have lost their ability to dimerize and, as a consequen
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