The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging

•A predicted structure for the T4 portal is proposed.•Mutagenesis of portal clip region residues identified five residues unable to be substituted.•The five residues include the proposed tunnel loop residue and all likely have important functions.•FRET determined a 7.5 nm distance between clip residue 316 and terminase C-terminus.•These studies support C-terminal nuclease domain docking of the terminase to the portal.

Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal-terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279-316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5 nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase-portal orientation fits better to a proposed “DNA crunching” compression packaging motor and to portal determined DNA headful cutting.