Genetic and biochemical analysis of dimer and oligomer interactions of thelambda S holin

Bacteriophage lambda uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane pr otein that permeabilizes the inner membrane at a precisely scheduled time a fter infection and allows the endolysin access to its substrate, resulting in host cell lysis. lambda S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from lambda S-C51S were tes ted for holin function. Most of the single-cysteine variants retained the a bility to support lysis. Mutations with the most defective phenotype cluste red in the first two hydrophobic transmembrane domains. Several lines of ev idence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge form ation, since the cysteineless S-C51S (i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contra st, the lysis-defective S-A52V mutant dimerizes but does not form cross-lin kable oligomers. Again, dimerization does not depend on the natural cystein e, since the cysteineless lysis-defective S-A52V/C51S is found in dimers af ter treatment of the membrane with a cross-linking agent. Furthermore, unde r oxidative conditions, dimerization via the natural cysteine is very effic ient for S-A52V. Both S-A52V (dominant negative) and S-A48V (antidominant) interact with the parental S protein, as judged by oxidative disulfide brid ge formation. Thus, productive and unproductive heterodimer formation betwe en the parental protein and the mutants S-A52V and S-A48V, respectively, ma y account for the dominant and antidominant lysis phenotypes. Examination o f oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that posi tions 48 and 51 are on a dimer interface. These results are discussed in te rms of a three-step model leading to S-dependent hole formation in the inne r membrane.