Protein Translocation Across Membranes: Components of Outer Membrane Colicin Translocons

Abstract

Translocation of the nuclease colicins E2 and E3 across the E. coli outer membrane is initiated by high affinity (Kd < 10−9 M) binding of the receptor-binding (R) domain to the vitamin B12 (BtuB) receptor in the E. coli outer membrane. Based on genetic analysis (1), and crystal structures of BtuB (2), the complex of the R-domain of colicins E2 or E3 bound to BtuB (3, 4), and of the OmpF porin containing the inserted N-terminal disordered segment of the colicin translocation (T) domain (5), a “fishing pole” model for the colicin translocon was inferred (3-5). The T- and C (catalytic) colicin segments must unfold before insertion into OmpF. FRET analysis was employed to study the colicin unfolding upon interaction with BtuB and OmpF (6). A rapid (k1/2 < 1 sec−1) decrease in FRET efficiency between translocation and cytotoxic domains of colicin E3 was observed upon independent and additive colicin binding in vitro to BtuB and OmpF. Colicin interactions with BtuB and OmpF have a major electrostatic component, provided at least partly for BtuB by R-domain Arg399. Thus, free energy for colicin unfolding is provided by binding of the R- domain to BtuB and also by binding/insertion of T-domain to OmpF. Screening the “Keio collection” for cytotoxicity of several group A and B colicins has shown thus far that colicin N binds to the first glucose of the LPS inner core (7).(1) Benedetti et al.,1989.(2) Cherezov et al., J. Mol. Biol, 364,716-, 2006.(3) Kurisu et al., Nat. Struct Biol., 10:948-, 2003.(4) Sharma et al. J. Biol.Chem., 282, 23163, 2007.(5) Yamashita et al., EMBO J., 27, 2171-, 2008.(6) Zakharov et al., Biochemistry, 47: in press, 2008.(7) Sharma et al., in preparation, 2008. [NIH-GM18457(WAC), NIH-GM083296(BLW)].