Structural Mechanism of Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Assembly in Lipid Bilayers Revealed by Solid-State NMR.

Synaptic vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including synaptobrevin-2 (Syb-2), syntaxin-1 (Syx-1), and SNAP-25. However, it remains controversial whether the formation of thoroughly contacted α-helical bundle from the SNARE motifs to the end of the transmembrane domains (TMDs) is necessary for SNARE-mediated membrane fusion. In this study, we characterized the conformation of Syb-2 in different assembly states using a combination of dipolar- and scalar-based solid-state NMR experiments in lipid bilayers. Our spectral analysis revealed a highly dynamic nature of the Syb-2 TMD with considerable α-helical contents. Chemical shift perturbation and mutational analysis indicated that the coupling between Syb-2 and Syx-1 TMDs mediated by residue Gly-100 of Syb-2 together with high mobility of the C-terminal segment of Syb-2 TMD are required for inner membrane merger. Our results provide new insights into the role of the Syb-2 TMD in driving membrane fusion, which improves the current understanding of the structural mechanism of SNARE complex assembly. This study highlights the significance of membrane environments in elucidating the mechanism of membrane proteins.

Dynamic Interactions Between Brilliant Green and MscL Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

The mechanosensitive ion channel of large conductance (MscL) is a promising template for the development of new antibiotics due to its high conservation and uniqueness to microbes. Brilliant green (BG), a triarylmethane dye, has been identified as a new antibiotic targeted MscL. However, the detailed binding sites to MscL and the dynamic pathway of BG through the MscL channel remain unknown. Here, the dynamic interactions between BG and MscL were investigated using solid-state NMR spectroscopy and molecule dynamics (MD) simulations. Residue site-specific binding sites of BG to the MscL channel were identified by solid-state NMR. In addition, MD simulations revealed that BG conducts through the MscL channel via residues along the inner surface of the pore sequentially, in which the strong hydrophobic interactions between BG and hydrophobic residues F23 and I27 in the hydrophobic gate region of the MscL channel are major restrictions. Particularly, it was demonstrated that BG activates the MscL channel by reducing the hydrophobicity of the F23 in the gate region by water molecules that are bound to BG. Taken together, these simulations and experimental data provide novel insights into the dynamic interactions between BG and MscL, based on which new hydrophobic antibiotics and adjuvants targeting MscL can be developed.

Molecular Mechanisms of Mercury-Sensitive Aquaporins.

Aquaporins are transmembrane channels that allow for the passive permeation of water and other small molecules across biological membranes. Their channel activities are sensitive to mercury ions. Intriguingly, while most aquaporins are inhibited by mercury ions, several aquaporins are activated by mercury ions. The molecular basis of the opposing aquaporin regulation by mercury remains poorly understood. Herein, we investigated AqpZ inhibition and AQP6 activation upon binding of mercury ions using solid-state NMR (ssNMR) and molecular dynamics (MD) simulations. Based on the structure of the Hg-AqpZ complex constructed by MD simulations and ssNMR, we identified that the pore closure was caused by mercury-induced conformational changes of the key residue R189 in the selectivity filter region, while pore opening was caused by conformational changes of residues H181 and R196 in the selectivity filter region in AQP6. Both conformational changes were caused by the disruption of the H-bond network of R189/R196 by mercury. The molecular details provided a structural basis for mercury-mediated functional changes in aquaporins.

Mechanism of unusual AQP6 activation by mercury binding to a pore-external residue C155.

Aquaporins (AQPs) transport water molecules across cell membranes. Although most aquaporins are inhibited by mercury ions, AQP6 was reported to be activated by binding mercury ions to residues C155 and C190. Different from C190 and the other pore-line cysteine residues, C155 is located outside the pore, thus not directly affecting the internal pathway by mercury binding to it. The molecular mechanism of unusual water channel activation by mercury ion binding to the C155 site remains unknown. Here, we investigate the activation of AQP6 by mercury ions binding to C155 by molecular dynamics (MD) simulations. The MD simulation results show that the mercury-induced water permeation activation is derived from the conformational change of a pore-line residue M160, from a point-to-pore conformation before mercury binding to an away-pore conformation after mercury binding. The conformation change of M160 is derived from the reduction of the hydrogen bonding between C155 and S159 in the α-helix with the coordination of C155 to mercury ion altering their conformation significantly. This study reveals the complex mechanism of water channel activation by mercury ion binding to pore-external residues in water channels.

Extensively sparse 13C labeling to simplify solid-state NMR 13C spectra of membrane proteins.

Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D 13C-13C spectra. Sparse labeling of 13C spins is an effective approach to simplify the 13C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein E. coli aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate 13C-12C labeling, and demonstrate an enhanced dilution effect in 13C-13C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate 13C-12C labeling). The signal intensities of some other types of residues in 2D 13C-13C spectra are observed to be 20-50% weaker because of the unwanted reverse labeling. The extensively sparse 13C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D 13C-13C spectra of membrane proteins.

Gating Mechanism of Aquaporin Z in Synthetic Bilayers and Native Membranes Revealed by Solid-State NMR Spectroscopy.

Aquaporin Z (AqpZ) is an integral membrane protein that facilitates transport of water across Escherichia coli cells with a high rate. Previously, R189, a highly conserved residue of the selective filter of AqpZ, was proposed as a gate within the water channel on the basis of the observation of both open and closed conformations of its side chain in different monomers of an X-ray structure, and the observation of rapid switches between the two conformations in molecular dynamic simulations. However, the gating mechanism of the R189 side chain remains controversial since it is unclear whether the different conformations observed in the X-ray structure is due to different functional states or is a result of perturbation of non-native detergent environments. Herein, in native-like synthetic bilayers and native E. coli membranes, a number of solid-state NMR techniques are employed to examine gating mechanism of the R189 side chain of AqpZ. One R189 side-chain conformation is highly evident since only a set of peaks corresponding to the R189 side chain is observed in 2D 15N-13C spectra. The immobility of the R189 side chain is detected by 1H-15N dipolar lineshapes, excluding the possibility of the rapid switches between the two side-chain conformations. High-resolution monomeric structure of AqpZ, determined by CS-Rosetta calculations using experimentally measured distance restraints related to the R189 side chain, reveals that this side chain is in an open conformation, which is further verified by its water accessibility. All the solid-state NMR experimental results, combining with water permeability essay, suggest a permanently open conformation of the R189 side chain in the synthetic bilayer and native membranes. This study provides new structural insights into the gating mechanism of aquaporins and highlights the significance of lipid bilayer environments in elucidating the molecular mechanism of membrane proteins.

Fast collective motions of backbone in transmembrane α helices are critical to water transfer of aquaporin.

Fast collective motions are widely present in biomolecules, but their functional relevance remains unclear. Herein, we reveal that fast collective motions of backbone are critical to the water transfer of aquaporin Z (AqpZ) by using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. A total of 212 residue site-specific dipolar order parameters and 158 15N spin relaxation rates of the backbone are measured by combining the 13C- and 1H-detected multidimensional ssNMR spectra. Analysis of these experimental data by theoretic models suggests that the small-amplitude (~10°) collective motions of the transmembrane α helices on the nanosecond-to-microsecond timescales are dominant for the dynamics of AqpZ. The MD simulations demonstrate that these collective motions are critical to the water transfer efficiency of AqpZ by facilitating the opening of the channel and accelerating the water-residue hydrogen bonds renewing in the selectivity filter region.

Solid-state NMR structure determination of a membrane protein in E. coli cellular inner membrane.

Structure determination of membrane proteins in native cellular membranes is critical to precisely reveal their structures in physiological conditions. However, it remains challenging for solid-state nuclear magnetic resonance (ssNMR) due to the low sensitivity and high complexity of ssNMR spectra of cellular membranes. Here, we present the structure determination of aquaporin Z (AqpZ) by ssNMR in Escherichia coli inner membranes. To enhance the signal sensitivity of AqpZ, we optimized protein overexpression and removed outer membrane components. To suppress the interference of background proteins, we used a "dual-media" expression approach and antibiotic treatment. Using 1017 distance restraints obtained from two-dimensional 13C-13C spectra based on the complete chemical shift assignments, the 1.7-Å ssNMR structure of AqpZ is determined in E. coli inner membranes. This cellular ssNMR structure determination paves the way for analyzing the atomic structural details for membrane proteins in native cellular membranes.

Membrane Protein Structures in Native Cellular Membranes Revealed by Solid-State NMR Spectroscopy.

The structural characterization of membrane proteins within the cellular membrane environment is critical for understanding the molecular mechanism in their native functional context. However, conducting residue site-specific structural analysis of membrane proteins in native membranes by solid-state NMR faces challenges due to poor spectral sensitivity and serious interference from background protein signals. In this study, we present a new protocol that combines various strategies for cellular membrane sample preparations, enabling us to reveal the secondary structure of the mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL) in Escherichia coli inner membranes. Our findings demonstrate the feasibility of achieving complete resonance assignments and the potential for determining the 3D structures of membrane proteins within cellular membranes. We find that the use of the BL21(DE3) strain in this protocol is crucial for effectively suppressing background protein labeling without compromising the sensitivity of the target protein. Furthermore, our data reveal that the structures of different proteins exhibit varying degrees of sensitivity to the membrane environment. These results underscore the significance of studying membrane proteins within their native cellular membranes when performing structural characterizations. Overall, this study opens up a new avenue for achieving the atomic-resolution structural characterization of membrane proteins within their native cellular membranes, providing valuable insights into the nativeness of membrane proteins.

Dynamics properties of membrane proteins in native cell membranes revealed by solid-state NMR spectroscopy.

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.

Solid-state NMR chemical shift assignments of aquaporin Z in lipid bilayers.

Aquaporin Z is the first identified prokaryotic water channel in Escherichia coli with a high water permeability and strict substrate selectivity. Here we report nearly complete (94% of amino acid residues) 13C and 15N chemical shift assignments of AqpZ reconstituted in the lipid bilayers using a set of 2D and 3D magic angle spinning solid-state NMR spectra. Secondary structure of AqpZ predicted from chemical shift assignments is generally similar to that of X-ray structure with a number of differences in loop and near-loop regions. The BMRB accession number of the assignments is 27244.

A robust heteronuclear dipolar recoupling method comparable to TEDOR for proteins in magic-angle spinning solid-state NMR.

In this letter, we propose a robust heteronuclear dipolar recoupling method for proteins in magic-angle spinning (MAS) solid-state NMR. This method is as simple, robust and efficient as the well-known TEDOR in the aspect of magnetization transfer between 15N and 13C. Deriving from our recent band-selective dual back-to-back pulses (DBP) (Zhang et al., 2016), this method uses new phase-cycling schemes to realize broadband DBP (Bro-DBP). For broadband 15N-13C magnetization transfer (simultaneous 15N→13C' and 15N→13Cα), Bro-DBP has almost the same 15N→13Cα efficiency while offers 30-40% enhancement on 15N→13C' transfer, compared to TEDOR. Besides, Bro-DBP can also be used as a carbonyl (13C')-selected method, whose 15N→13C' efficiency is up to 1.7 times that of TEDOR and is also higher than that of band-selective DBP. The performance of Bro-DBP is demonstrated on the N-formyl-[U-13C,15N]-Met-Leu-Phe-OH (fMLF) peptide and the U-13C, 15N labeled ß1 immunoglobulin binding domain of protein G (GB1) microcrystalline protein. Since Bro-DBP is as robust, simple and efficient as TEDOR, we believe it is very useful for protein studies in MAS solid-state NMR.