Mutational analysis of the S21 pinholin.
Lambdoid phage 21 has the prototype pinholin-SAR endolysin lysis system, which is widely distributed among phages. Its prototype pinholin, S(21)68, triggers at an allele-specific time to form small, heptameric lesions, or pinholes, in the cytoplasmic membrane, thus initiating lysis. S(21)68 has two transmembrane domains, TMD1 and TMD2. Only TMD2 is required for the formation of pinholes, whereas TMD1 acts as an inhibitor of TMD2 and must be externalized to the periplasm in the lytic pathway. Previously we provided evidence that S(21)68 first accumulates as inactive dimers with both transmembrane domains embedded in the bilayer. Here we analyse an extensive collection of S(21) mutants to identify residues and domains critical to the function and regulation of the pinholin. Evidence is presented indicating that, within the inactive dimer, TMD1 acts in trans as an inhibitor of the lethal function of TMD2. A wide range of phenotypes, from absolute lysis defectives to accelerated lysis triggering, are observed for mutations mapping to each topological domain. The pattern of phenotypes allows the generation of a model for the structure of the inactive dimer. The model identifies the faces of the two transmembrane domains involved in intramolecular and intermolecular interactions, as well as interaction with the lipid.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Ting
T
Pang
Taehyun
T
Park
Ry
R
Young
D001433:Bacteriolysis; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017906:Siphoviridae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D014764:Viral Proteins; D010450:Endopeptidases; C001143:endolysin