Molecular Basis of Tetramerization and pH-Gating in the KcsA Potassium Channel Cytoplasmic Domain

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The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue long segment which natively adopts a helical bundle conformation with four-fold symmetry. A hallmark of KcsA behavior is a pH-induced conformational change which leads to opening of the channel at acidic pH. While crystal structures of full-length KcsA failed to observe a pH-effect upon the CTD, other biophysical methods have presented evidence to the contrary. We approached the question of CTD structure and its pH-dependence by studying the behavior of soluble peptides corresponding to residues 128-160 of the CTD (CTD34). A combination of NMR and sedimentation equilibrium experiments established CTD34 to be a tetramer with a KD of (2.0 ± 0.5) x 10−11 M3 at neutral pH, and this tetrameric species undergoes pH-dependent dissociation, rendering CTD34 fully monomeric below pH 5.0. The structural basis for this phenomenon is destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide.Molecular factors contributing to CTD tetramerization were investigated by comparing the tetrameric stability of single alanine mutants as determined by NMR, SE and molecular dynamics. Single-residue contributions to tetramer stability were in the 0.5-3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than the inter-subunit salt-bridges between R147 and D149/E152. A third class of residues outside the helical interface influenced tetramer stability via the tetramerization on-rate by changing the inherent helical propensity of CTD34 which promotes tetramer formation. We conclude that the CTD is an independent tetramerization domain modulated by pH, and tetramer formation is controlled by a combination of on- and off-rate effects which conform to current paradigms of protein folding.