Resolving the native conformation of Escherichia coli OmpA.

The native conformation of the 325-residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow-pore, two-domain structure has vied with a large-pore, single-domain structure. Our recent studies show that Ser163 and Ser167 of the N-terminal domain (1-170) are modified in the cytoplasm by covalent attachment of oligo-(R)-3-hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N-terminal domain to be incorporated into planar lipid bilayers as narrow pores (≈ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M-OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I-OmpA). Chemical and Western blot analysis and 1H-NMR showed that segment 264-325 in M-OmpA, but not in I-OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA. Planar lipid bilayer studies indicated that narrow pores formed by M-OmpA undergo a temperature-induced transition into stable large pores (≈ 450 pS, 1 M KCl, 22 °C) [energy of activation (Ea) = 33.2 kcal·mol(-1)], but this transition does not occur with I-OmpA or with M-OmpA that has been exposed to disulfide bond-reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB-modification, disulfide bond formation and temperature in folding OmpA into its native large-pore configuration.

Importance of oligo-R-3-hydroxybutyrates to S. lividans KcsA channel structure and function.

In the polyphosphate model of the Streptomyces lividans potassium channel KcsA, four polypeptides, each covalently modified by oligo-(R)-3-hydroxybutyrates (cOHB), surround a core molecule of inorganic polyphosphate (polyP). PolyP attracts, binds, and conducts K(+) in response to an electrochemical stimulus whilst the polypeptides govern access to polyP and regulate its selectivity. However, the role of cOHB has remained uncertain. Here we identify cOHB-conjugated residues in the ion pathway, S102 and S129, and mutate them to determine the influence of cOHB on channel properties. We find that the mutations have no discernible effect on tetramer formation or tetramer stability; however, cOHB influences polyP incorporation and/or retention, i.e. single mutants S102G and S129G contain ∼1/3 and double mutant S102G:S129G ≈ 1/2 as much polyP as wild-type. Moreover, planar lipid bilayer studies of wild-type and mutant proteins indicate that cOHB has a critical effect on channel function: at positive potentials, only ∼5% of S102G and S129G currents and <1% of S102G:S129G currents consist of well-structured channels; at negative potentials, S102G and S129G display only irregular conductance and S102G:S129G exhibits no conductance whatsoever. The results indicate that cOHB facilitates the incorporation and/or retention of polyP and plays a critical role in maintaining the flexible polyP molecule in an optimal transbilayer orientation for efficient K(+) transport.

Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel.

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity-lysine, histidine, valine, asparagine-and the effects of these mutations on conductance and selectivity for K(+) over Mg(2+) is tested in planar bilayers as a function of Mg(2+) concentration and pH. As the basicity of the C-terminal residues decreases, Mg(2+) block increases, and Mg(2+) becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.