Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel

Abstract

As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

High-resolution magic angle spinning NMR of KcsA in liposomes: the highly mobile C-terminus

“…Protein-free liposomes were also examined to confirm the resonances arising from lipids. From this comparison, we concluded that much of the protein signal in spectra of the full- [3,23,26,27] or Baldus Group ( †) [25,28,29]. Structural regions are indicated to the right of residue numbers.…”

“…Figure 1. Solid-state NMR backbone assignments of KcsA in liposomes as described by the McDermott Group (*)[3,23,26,27] or Baldus Group ( †)[25,28,29]. Structural regions are indicated to the right of residue numbers.…”

TUPÃ: Electric field analyses for molecular simulations

“…Ion channels play crucial roles in biological processes and have been thoroughly studied via MD simulations to better comprehend the molecular forces driving ion transport, flux, and selectivity. [36][37][38][39][40][41] Here, we studied the double mutant (D66Y and N68D) NaK2K reported by Derebe et al 28 and derived from the NaK channel family to demonstrate the electric field organization throughout the channel core.…”

Reassessing the helix bundle crossing model for gating in a non-selective ion channel

“…Meanwhile, solid and solution-state NMR studies on KcsA and KirBac1.1 have interpreted chemical shift perturbation data as supporting the two state HBC gating model [10][11][12][13][14][15] . However, in the case of KirBac3.1 it was later demonstrated that inner helix bundle stabilized with disulfide bonds in a closed conformation still allows K + ion conduction 16 and NMR chemical shift guided structural investigation of the conductive conformation of KcsA suggested a much narrower HBC opening than initially believed from the X-ray structures 17 . The struggle of assigning structures with different HBC conformations to functional states continues with more recent EM structures, as exemplified by the TASK channel 18,19 , where structural interpretations do not necessarily agree with the functional data and a residue far away from HBC appears to be critical for gating 20 .…”

“…Unlike solid-state NMR 43 spectra of NaK in liposomes, solution state NMR spectra of NaK samples in lipid bicelles display clear resonances for residues throughout the M 2 helix, including those near the HBC 44 . Additionally, while substantial attention has been paid to investigating KcsA by both solution and solid-state NMR, these studies largely excluded detailed investigation of the HBC, only indirectly correlating chemical shift perturbation data with hypothesized changes in the structure 12,14,17 : in part due to the ambiguity of the detailed atomic structures of different states 17 . While a powerful technique for unraveling structure and dynamics in smaller proteins, for large molecular systems such as ion channels, obtaining the high quality NMR data necessary for meaningful interpretation can be a challenge.…”