The extreme C-terminal region of the phage BFK20 gp41 helicase has a role in DNA binding, protein-ATP interactions and ATPase activity.

Replication protein gp41 from bacteriophage BFK20 is a 537 residue SF2 family helicase. The N-terminal two-thirds of the gp41 sequence is homologous to XPB/Ssl2-like helicases, but no clear homology to any known and characterized protein could be found for the C-terminal one-third. We prepared and studied the following gp41 mutant recombinant proteins: deletion mutant gp41L481, missing the last 56 C-terminal amino acids (482-537), and five point mutants, each substituting a single amino acid from this region with alanine (K516A, R518A, D520A, D521A and E522A). We tested the ATPase activities, DNA binding abilities, thermal stabilities and protein-ATP interactions of each isolated protein and compared them with wild-type-like protein gp41HN. The ATPase activity and DNA binding ability of gp41L481 were significantly lower than gp41HN. The K516A and R518A mutations resulted in an almost total loss of ATPase activity, while the D521A mutation produced a lesser loss. The K516A mutation also significantly reduced the DNA binding ability of the mutant protein. All point mutants were less stable than the wild-type protein to a greater or lesser extent, and ATP had a significant stabilizing effect on most tested proteins. We conclude that the amino-acids at the extreme C-terminus of gp41 are important for its ATPase activity, DNA binding ability and protein-ATP interactions. BFK20 gp41 is an example of a phage helicase whose accessory domain significantly affects its properties and it provides additional evidence for the importance of accessory domains for helicase function.