Spanin function requires subunit homodimerization through intermolecular disulfide bonds

Summary The λ R z and R z1 proteins are the subunits of the spanin complex, required for the disruption of the outer membrane during host lysis. R z, the inner membrane or i‐spanin, has a largely alpha‐helical periplasmic domain, whereas R z1, the outer membrane or o‐spanin, has a 25% proline content with no predicted secondary structure. We report that both R z and R z1 accumulate as homodimers covalently linked by intermolecular disulfide bonds involving all three C ys residues, two in R z and one in R z1. Moreover, of these three intermolecular disulfides, spanin function requires the presence of at least one of the two linkages nearest the R z– R z1 C ‐terminal interaction domains; i.e. either the R z1– R z1 disulfide or the distal R z– R z disulfide link. In a dsbC host, but not in dsbA or dsbA dsbC hosts, formation of the covalent homodimers of R z is severely reduced and outer membrane disruption is significantly delayed, suggesting that the spanin pathway normally proceeds through DsbA ‐mediated formation of an intramolecular disulfide in R z. In contrast, efficient formation of the R z1– R z1 disulfide requires DsbA . Finally, D sb‐independent formation of the covalent homodimer of either subunit requires the presence of the other, presumably as a template for close apposition of the thiols.