Symmetry-Constrained Analysis of Pulsed Double Electron–Electron Resonance (DEER) Spectroscopy Reveals the Dynamic Nature of the KcsA Activation Gate

Abstract

Distance determination from an echo intensity modulation obtained by pulsed double electron-electron resonance (DEER) experiment is a mathematically ill-posed problem. Tikhonov regularization yields distance distributions that can be difficult to interpret, especially in system with multiple discrete distance distributions. Here, we show that by using geometric fit constraints in symmetric homo-oligomeric protein systems, we were able to increase the accuracy of a model-based fit solution based on a sum of Gaussian distributions. Our approach was validated on two different ion channels of known oligomeric states, KcsA (a tetramer) and CorA (a pentamer). Statistical analysis of the resulting fits was integrated within our method to help the experimenter evaluate the significance of a symmetry-constrained vs. standard model distribution fit and to examine multi-distance confidence regions. This approach was used to quantitatively evaluate the role of the C-terminal domain (CTD) on the flexibility and conformation of the activation gate of the K+ channel KcsA. Our analysis reveals a significant increase in the dynamics of the inner bundle gate upon opening. Also, it explicitly demonstrates the degree to which the CTD restricts the motion of the lower gate at rest and during activation gating.

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

“…(14). In line with electron paramagnetic resonance data (52,53), our results show that the transition from neutral to acidic pH is accompanied by a gradual closing of the inactivation gate and an increasing opening of the activation gate. According to our ssNMR data, two channel states exist with a partially open activation gate and open and closed inactivation gates in CL bilayers at pH 4.…”

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

“…The DEER experiment has shown to be a valuable technique to measure inter-spin distances within membrane proteins (Dellisanti et al, 2013; Jeschke, 2012; Jeschke et al, 2004). In particular, for a pentameric assembly containing single-labeled subunits, a bimodal distribution (Figure 5a and 5b) consisting of an adjacent and non-adjacent distance is expected (Dalmas et al, 2012). For a pentagon, a distance ratio of ~1.6 between the non-adjacent and adjacent sites is anticipated (Figure 5c).…”

Conformational Cycle of the Vitamin B12 ABC Importer in Liposomes Detected by Double Electron-Electron Resonance (DEER)

“…DEER is a powerful tool to reveal functional intermediate states in ABC transporters (8, 16 -20), symporters (21), or ion channels (22,23), and it can be applied in a native-like lipid environment providing inter-residue distance information in the 1.5-6-nm range (24). The BtuCD-F translocation channel consists of residues from TM5-TM5a helices, the loop between TM2 and TM3 (cytoplasmic gate II) and a few residues from TM3 and TM8 helices; the channel is further flanked with TM10 residues (Fig.…”