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

Journal: Journal of structural biology 2021;210(3):107508

PMID: 32298813

Full Text on Elsevier Science View on PubMed

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.

Address: Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia. Electronic address: [email protected].; Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.; Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.; Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria.; European Synchrotron Radiation Facility, Grenoble 38000, France.

DOI: 10.1016/j.jsb.2020.107508

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MeSH Terms: Catalytic Domain, Deoxyribonuclease I, Mutagenesis, Site-Directed, Mutation