The key role of E418 carboxyl group in the formation of Nt.BspD6I nickase active site: Structural and functional properties of Nt.BspD6I E418A mutant

Rimma I Artyukh; Galina S Kachalova; Alfiya K Yunusova; Bulat F Fatkhullin; Boris P Atanasov; Tatyana A Perevyazova; Alexander N Popov; Azat G Gabdulkhakov; Ludmila A Zheleznaya

doi:10.1016/j.jsb.2020.107508

The key role of E418 carboxyl group in the formation of Nt.BspD6I nickase active site: Structural and functional properties of Nt.BspD6I E418A mutant

J Struct Biol. 2020 Jun 1;210(3):107508. doi: 10.1016/j.jsb.2020.107508. Epub 2020 Apr 13.

Authors

Rimma I Artyukh¹, Galina S Kachalova², Alfiya K Yunusova², Bulat F Fatkhullin³, Boris P Atanasov⁴, Tatyana A Perevyazova², Alexander N Popov⁵, Azat G Gabdulkhakov³, Ludmila A Zheleznaya²

Affiliations

¹ Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia. Electronic address: rimmaartyukh@gmail.com.
² Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
³ Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
⁴ Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria.
⁵ European Synchrotron Radiation Facility, Grenoble 38000, France.

PMID: 32298813
DOI: 10.1016/j.jsb.2020.107508

Abstract

The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.

Keywords: Cleavage domain; Crystal structure; Nickase; Recognition domain; Site-directed mutagenesis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Catalytic Domain / genetics
Deoxyribonuclease I / chemistry*
Deoxyribonuclease I / genetics
Deoxyribonuclease I / metabolism*
Mutagenesis, Site-Directed
Mutation / genetics

Substances

Deoxyribonuclease I