Structural and functional insights into Gp21 as a new SF4 helicase of prolate-headed Lactococcus lactis phage 94p4

Abstract

The Gp21 protein is encoded in the early gene region of the lytic, prolate-headed (Ceduovirus type) Lactococcus lactis bacteriophage 94p4 genome. By in silico modelling we found that the protein shares significant structural and motif-specific homology with superfamily 4 (SF4) replicative helicases, such as Escherichia coli DnaB, phage T4 Gp41, and phage T7 Gp4. Our study demonstrates that Gp21 possesses robust DNA unwinding activity, efficiently separating strands of DNA heteroduplexes in a 5′ to 3′ direction. Biochemical characterization revealed that Gp21 forms hexamers and requires ATP and Mg2+ as cofactors for optimal activity. Site-directed mutagenesis of conserved residues within Gp21 impaired both its unwinding activity and hexamer formation, further supporting its classification as an SF4 helicase. The functional and structural similarity of Gp21 to SF4 replicative DNA helicases strongly suggests its role in DNA replication. This discovery identifies Gp21 as the first functionally confirmed SF4 helicase in Ceduovirus phages, offering new insights into the replication of this phage family.