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.
Negoda
Alexander
A
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Negoda
Elena
E
Reusch
Rosetta N
RN
D020155:3-Hydroxybutyric Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015640:Ion Channel Gating; D007477:Ions; D007525:Isoelectric Focusing; D008051:Lipid Bilayers / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D048170:Streptomyces lividans / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D007477:Ions; D008051:Lipid Bilayers; D050505:Mutant Proteins; D011122:Polyphosphates; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D020155:3-Hydroxybutyric Acid