Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization.

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states.

Beyond structure: Deciphering site-specific dynamics in proteins from double histidine-based EPR measurements.

Site-specific dynamics in proteins are at the heart of protein function. While electron paramagnetic resonance (EPR) has potential to measure dynamics in large protein complexes, the reliance on flexible nitroxide labels is limitating especially for the accurate measurement of site-specific ß-sheet dynamics. Here, we employed EPR spectroscopy to measure site-specific dynamics across the surface of a protein, GB1. Through the use of the double Histidine (dHis) motif, which enables labeling with a Cu(II) - nitrilotriacetic acid (NTA) complex, dynamics information was obtained for both α-helical and ß-sheet sites. Spectral simulations of the resulting CW-EPR report unique site-specific fluctuations across the surface of GB1. Additionally, we performed molecular dynamics (MD) simulations to complement the EPR data. The dynamics observed from MD agree with the EPR results. Furthermore, we observe small changes in gǁ values for different sites, which may be due to small differences in coordination geometry and/or local electrostatics of the site. Taken together, this work expands the utility of Cu(II)NTA-based EPR measurements to probe information beyond distance constraints.

Allostery-driven changes in dynamics regulate the activation of bacterial copper transcription factor.

Metalloregulators bind and respond to metal ions by regulating the transcription of metal homeostasis genes. Copper efflux regulator (CueR) is a copper-responsive metalloregulator that is found in numerous Gram-negative bacteria. Upon Cu(I) coordination, CueR initiates transcription by bending the bound DNA promoter regions facilitating interaction with RNA polymerase. The structure of Escherichia coli CueR in presence of DNA and metal ion has been reported using X-ray crystallography and cryo-EM, providing information about the mechanism of action. However, the specific role of copper in controlling this transcription mechanism remains elusive. Herein, we use room temperature electron paramagnetic resonance (EPR) experiments to follow allosterically driven dynamical changes in E. coli CueR induced by Cu(I) binding. We suggest that more than one Cu(I) ion binds per CueR monomer, leading to changes in site-specific dynamics at the Cu(I) binding domain and at the distant DNA binding site. Interestingly, Cu(I) binding leads to an increase in dynamics about 27 Å away at the DNA binding domain. These changes in the dynamics of the DNA binding domain are important for exact coordination with the DNA. Thus, Cu(I) binding is critical to initiate a series of conformational changes that regulate and initiate gene transcription. BROAD AUDIENCE STATEMENT: The dynamics of metal transcription factors as a function of metal and DNA binding are complex. In this study, we use EPR spectroscopy to measure dynamical changes of Escherichia coli CueR as a function of copper and DNA binding. We show that copper controls the activation of the transcription processes by initiation a series of dynamical changes over the protein.

Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

Cleavage-Resistant Protein Labeling With Hydrophilic Trityl Enables Distance Measurements In-Cell.

Sensitive in-cell distance measurements in proteins using pulsed-electron spin resonance (ESR) require reduction-resistant and cleavage-resistant spin labels. Among the reduction-resistant moieties, the hydrophilic trityl core known as OX063 is promising due to its long phase-memory relaxation time (Tm). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the Tm of OX063 remains sufficiently long at higher temperatures, opening the possibility for measurements at temperatures above 50 K. In this work, we synthesized deuterated OX063 with a maleimide linker (mOX063-d24). We show that the combination of the hydrophilicity of the label and the maleimide linker enables high protein labeling that is cleavage-resistant in-cells. Distance measurements performed at 150 K using this label are more sensitive than the measurements at 80 K. The sensitivity gain is due to the significantly short longitudinal relaxation time (T1) at higher temperatures, which enables more data collection per unit of time. In addition to in vitro experiments, we perform distance measurements in Xenopus laevis oocytes. Interestingly, the Tm of mOX063-d24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d24 provides highly sensitive distance measurements both in vitro and in-cells.

Going the dHis-tance: Site-Directed Cu2+ Labeling of Proteins and Nucleic Acids.

In this Account, we showcase site-directed Cu2+ labeling in proteins and DNA, which has opened new avenues for the measurement of the structure and dynamics of biomolecules using electron paramagnetic resonance (EPR) spectroscopy. In proteins, the spin label is assembled in situ from natural amino acid residues and a metal complex and requires no post-expression synthetic modification or purification procedures. The labeling scheme exploits a double histidine (dHis) motif, which utilizes endogenous or site-specifically mutated histidine residues to coordinate a Cu2+ complex. Pulsed EPR measurements on such Cu2+-labeled proteins potentially yield distance distributions that are up to 5 times narrower than the common protein spin label-the approach, thus, overcomes the inherent limitation of the current technology, which relies on a spin label with a highly flexible side chain. This labeling scheme provides a straightforward method that elucidates biophysical information that is costly, complicated, or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at ß-sheet sites, which are largely inaccessible through traditional spin labels, and rigid Cu2+-Cu2+ distance measurements that enable higher precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. Likewise, a Cu2+ label has been developed for use in DNA, which is small, is nucleotide independent, and is positioned within the DNA helix. The placement of the Cu2+ label directly reports on the biologically relevant backbone distance. Additionally, for both of these labeling techniques, we have developed models for interpretation of the EPR distance information, primarily utilizing molecular dynamics (MD) simulations. Initial results using force fields developed for both protein and DNA labels have agreed with experimental results, which has been a major bottleneck for traditional spin labels. Looking ahead, we anticipate new combinations of MD and EPR to further our understanding of protein and DNA conformational changes, as well as working synergistically to investigate protein-DNA interactions.

Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy.

Site-directed spin labeling in conjunction with electron paramagnetic resonance (EPR) is an attractive approach to measure residue specific dynamics and point-to-point distance distributions in a biomolecule. Here, we focus on the labeling of proteins with a Cu(II)-nitrilotriacetic acid (NTA) complex, by exploiting two strategically placed histidine residues (called the dHis motif). This labeling strategy has emerged as a means to overcome key limitations of many spin labels. Through utilizing the dHis motif, Cu(II)NTA rigidly binds to a protein without depending on cysteine residues. This protocol outlines three major points: the synthesis of the Cu(II)NTA complex; the measurement of continuous wave and pulsed EPR spectra, to verify a successful synthesis, as well as successful protein labeling; and utilizing Cu(II)NTA labeled proteins, to measure distance constraints and backbone dynamics. In doing so, EPR measurements are less influenced by sidechain motion, which influences the breadth of the measured distance distributions between two spins, as well as the measured residue-specific dynamics. More broadly, such EPR-based distance measurements provide unique structural constraints for integrative structural biophysics and complement traditional biophysical techniques, such as NMR, cryo-EM, FRET, and crystallography. Graphic abstract: Monitoring the success of Cu(II)NTA labeling.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site-Specific Elucidation of Hidden Dynamics in Enzymes.

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-CuII NTA label to obtain site-specific information on dynamics for both an α-helix and ß-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure site-specific dynamics using room temperature lineshape analysis.

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution.

Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2'dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

19F Paramagnetic Relaxation-Based NMR for Quaternary Structural Restraints of Ion Channels.

Quaternary distance restraints are essential to define the three-dimensional structures of protein assemblies. These distances often fall within a range of 10-18 Å, which challenges the high and low measurement limits of conventional nuclear magnetic resonance (NMR) and double electron-electron resonance electron spin resonance spectroscopies. Here, we report the use of 19F paramagnetic relaxation enhancement (PRE) NMR in combination with 19F/paramagnetic labeling to equivalent sites in different subunits of a protein complex in micelles to determine intersubunit distances. The feasibility of this strategy was evaluated on a pentameric ligand-gated ion channel, for which we found excellent agreement of the 19F PRE NMR results with previous structural information. The study suggests that 19F PRE NMR is a viable tool in extracting distance restraints to define quaternary structures.

Increasing nitroxide lifetime in cells to enable in-cell protein structure and dynamics measurements by electron spin resonance spectroscopy.

There is increasing evidence that the stability, structure, dynamics, and function of many proteins differ in cells versus in vitro. The determination of protein structure and dynamics within the native cellular environment may lead to better understanding of protein behavior. Electron spin resonance (ESR) has emerged as a technique that can report on protein structure and dynamics within cells. Nitroxide based spin labels are capable of reporting on protein dynamics, structure, and backbone flexibility but are limited due to nitroxide reduction occurring in cells. In order to overcome this limitation, we used the oxidizing agent potassium ferricyanide (K3Fe(CN)6) as well as the cleavage resistant spin label 3-malemido-PROXYL (5-MSL). Furthermore, we hypothesized that injection concentration is an important parameter regarding nitroxide reduction kinetics. By increasing the injection concentration of doubly 5-MSL labeled protein into Xenopus laevis oocytes, we found an increased nitroxide lifetime. Our work demonstrates unprecedented incubation times of 3-h in-cell and 5-h in-cytosol for double electron-electron resonance (DEER) experiments using nitroxide spin labels. This allows for more meaningful measurements of larger protein systems which may require longer incubation times for equilibration in the cellular milieu. Even longer incubation times are possible by combining our approach with more shielded nitroxides and Q-band.