A dimeric holin/antiholin complex controls lysis by phage T4

Lytic phages control the timepoint of host cell lysis by timing the holin-mediated
release of cell wall-degrading endolysins. In phage T4, the antiholin RI inhibits
the holin T, thereby preventing the early release of the T4 endolysin and lysis.
The antiholin achieves lysis inhibition (LIN) in response to phage superinfections,
thereby increasing the chance for lysis in an environment with a lower phage
concentration. The holin T consists of a small N-terminal cytoplasmic domain,
a transmembrane helix, and a periplasmic C-terminal domain. The antiholin is
targeted to the periplasm by a cleavable signal peptide. Recently, the periplasmic
soluble domains of the holin and the antiholin were found to form T2/RI2
tetramers in crystals. To investigate the functional relevance of this complex,
we reconstituted LIN in a phage-free system, using only RI, T, and endolysin,
and combined targeted mutagenesis with functional analyses. Inactivation of
the RI signal peptide cleavage site did not abolish LIN, indicating that RI can
function in a membrane-bound state, which argued against the tetramer. This
led to analyses showing that only one of the two T/RI interfaces in the tetramer
is physiologically relevant, which is also the only interaction site predicted
by AlphaFold2. Some holin mutations at this interaction site prevented lysis,
suggesting that the RI interaction likely acts by blocking the holin oligomerization required for hole formation. We conclude that LIN is mediated by a dimeric T/RI complex that, unlike the tetramer, can be easily formed when both partners are membrane-anchored.