Molecular determinants of tetramerization in the KcsA cytoplasmic domain. (12th August 2014)

• Record Type: Journal Article
• Authors: Kamnesky, Guy; Hirschhorn, Orel; Shaked, Hadassa; Chen, Jingfei; Yao, Lishan; Chill, Jordan H.
• Is Part Of: Protein science. Volume 23:Number 10(2014:Oct.)
• Page Start: 1403
• Page End: 1416
• Publication Date: 2014-08-12
• Subjects: Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.2525
• Languages: English
• ISSNs: 0961-8368

Abstract

<abstract abstract-type="main"> <title>Abstract</title> <p>The cytoplasmic C‐terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four‐fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD‐derived 34‐residue peptide associates into a tetramer in a pH‐dependent manner (Kamnesky et al., JMB 2012;418:237‐247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer‐tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single‐residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter‐subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter‐subunit interface also influenced tetramer stability by affecting the tetramerization on‐rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein‐protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full‐length KcsA channels.</p> </abstract>