Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy

Abstract

Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNDB) of the HCN2 ion channel upon binding of the ligand 3′,5′-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC+normalL⇌AO+normalL⇌BO⇌BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

DeerLab: A comprehensive toolbox for analyzing dipolar EPR spectroscopy data

“…Such titration or dose-response datasets have been analyzed using multi-Gaussian distribution models for the component distributions (Stein et al, 2015;Martens et al, 2016;Collauto et al, 2017;Barth et al, 2018;Jagessar et al, 2020). As discussed above, however, parameter-free distributions may often be a preferable choice.…”

Multimerization of Homo sapiens TRPA1 ion channel cytoplasmic domains

“…This is just one way in which the behavior of isolated channel fragments in solution may not faithfully represent their behavior in the intact protein when embedded in a lipid bilayer. Nevertheless, studying isolated fragments in solution has revealed mechanistic insights in cys-loop channels [40], glutamate receptor channels [41,42], Ca 2+ -activated K + channels [43,44], cyclic nucleotide-binding domains of HCN channels [45–47], and others. It is encouraging that the recombinant coiled-coil in our system bound the ARD, as observed in cryoEM studies of TRPA1.…”

A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

“…For a homo-spin pair there is a trade-off between measurement sensitivity and the accuracy of the observed modulation depth quotients, (S TMU ), depending on the error in the approximated T1 value. Importantly, the largest source of error will likely manifest as deviations from the mono-exponential approximation, shown in equation (30). RIDME measurements were performed with 3 experimental mixing times, since the observed modulation depth is a function of both dH loading and ∆ KL M .…”

“…[13][14][15][16] PDEPR has contributed to conformational studies, [17][18][19] disentangling competing structural models, [20,21] and provided mechanistic insights into complex biomolecular apparatus. [22][23][24] Furthermore, PDEPR has been used to monitor complexation, [25][26][27] determine solution-state protein-ligand binding equilibria, [28][29][30] and study oligomerisation-degree. [31][32][33][34] Commonly, pairs of paramagnetic moieties, such as nitroxide radicals, are covalently introduced and conjugated with thiol side-chains of cysteine residues inserted at strategic positions via site-directed mutagenesis.…”