Magnetically Aligned Lipid Bilayers with High Cholesterol for Solid-State NMR of Membrane Proteins.

Phospholipid bicelles are valuable membrane model systems to study membrane proteins by NMR and other physicochemical techniques. The range of bicelle compositions that are compatible with uniaxial alignment of the lipid bilayers in a magnetic field is still limited with regard to the addition of large amounts (>20%) of cholesterol and/or sphingolipids. Here, we demonstrate that n-dodecyl-ß-D-melibioside (DDMB), which was recently introduced as a detergent to produce sphingolipid-cholesterol-rich isotropic bicelles for solution NMR studies, can also be used to produce magnetically alignable lipid bilayers with high cholesterol content that are well suited for solid-state NMR of membrane proteins. Remarkably, DDMB enables the preparation of high q bicelles that contain 50% mol cholesterol while retaining their ability to form a stable, well-aligned liquid crystalline bilayer phase in a magnetic field. We show that the intact 46-residue membrane-bound form of Pf1 bacteriophage coat protein and a truncated construct of the membrane protein Vpu from HIV-1 (residues 2-30) in DDMB bicelles are well aligned and undergo fast and uniaxial rotational diffusion about the bilayer normal, similarly to what is observed in other bicelle and macrodisc systems. We also demonstrate a spectroscopic method that measures the increase in the thickness of DMPC bilayers that results from the addition of cholesterol, using the PISA-wheel spectral patterns of trans-membrane helices as a molecular goniometer. For example, we find that the hydrophobic thickness of DMPC bilayers is increased by approximately 2.5 Å in the presence of 35% mol cholesterol.

Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity.

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

3D printed sample tubes for solid-state NMR experiments.

The availability of 3D printers and an assortment of polymers that can be fashioned into a wide variety of shapes provides opportunities to rethink the design and construction of probes for NMR spectroscopy. The direct interfacing of computer aided design (CAD) with precise 3D printing enables the simplification and optimization of probes through the rapid production of components. Here we demonstrate the use of 3D printing to fully integrate a permanent former for the radiofrequency (RF) coil with the sample chamber (equivalent to the sample tube). This simultaneously increases the sample volume and improves the filling factor within a fixed outer diameter (OD). It also reduces the space lost in dual coil arrangements where a high frequency resonator is positioned outside a solenoid coil tuned to one or more lower frequencies, making multiple-resonance experiments more efficient. The initial applications demonstrate the possibilities for future designs that reimagine the interface between resonators and the liquid, solid, and heterogeneous samples encountered in NMR studies of biomolecules, polymers, surfaces, and spectroscopy (MRS) and imaging (MRI) of biological organs and intact organisms.

Correlating the Structure and Activity of Y. pestis Ail in a Bacterial Cell Envelope.

Understanding microbe-host interactions at the molecular level is a major goal of fundamental biology and therapeutic drug development. Structural biology strives to capture biomolecular structures in action, but the samples are often highly simplified versions of the complex native environment. Here, we present an Escherichia coli model system that allows us to probe the structure and function of Ail, the major surface protein of the deadly pathogen Yersinia pestis. We show that cell surface expression of Ail produces Y. pestis virulence phenotypes in E. coli, including resistance to human serum, cosedimentation of human vitronectin, and pellicle formation. Moreover, isolated bacterial cell envelopes, encompassing inner and outer membranes, yield high-resolution solid-state NMR spectra that reflect the structure of Ail and reveal Ail sites that are sensitive to the bacterial membrane environment and involved in the interactions with human serum components. The data capture the structure and function of Ail in a bacterial outer membrane and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum.

1H detection of heteronuclear dipolar oscillations with water suppression in single crystal peptide and oriented protein samples.

Oriented sample solid-state NMR is a complementary approach to protein structure determination with the distinct advantage that it can be applied to supramolecular assemblies, such as viruses and membrane proteins, under near-native conditions, which generally include high levels of hydration as found in living systems. Thus, in order to perform 1H detected versions of multi-dimensional experiments water suppression techniques must be integrated into the pulse sequences. For example, 1H-windowed detection of 1H-15N dipolar couplings enable multi-dimensional NMR experiments to be performed. Here we show that the addition of a solvent suppression pulse during the z-filter interval greatly improves the sensitivity of the experiments by suppressing the 1H signals from water present. This is demonstrated here with a crystal sample submerged in water and then extended to proteins. The combination of solvent-suppressed 1H detected PISEMO and the use of a strip shield-solenoid coil probe configuration provides a two-fold sensitivity enhancement in both the crystal sample and Pf1 coat protein sample compared to the 15N direct detection method. Here we also examine protein NMR line-widths and sensitivity enhancements in the context of window detected separated local field experiments for protein samples.

Membrane proteins in magnetically aligned phospholipid polymer discs for solid-state NMR spectroscopy.

Well-hydrated phospholipid bilayers provide a near-native environment for membrane proteins. They enable the preparation of chemically-defined samples suitable for NMR and other spectroscopic experiments that reveal the structure, dynamics, and functional interactions of the proteins at atomic resolution. The synthetic polymer styrene maleic acid (SMA) can be used to prepare detergent-free samples that form macrodiscs with diameters greater than 30 nm at room temperature, and spontaneously align in the magnetic field of an NMR spectrometer at temperatures above 35 °C. Here we show that magnetically aligned macrodiscs are particularly well suited for solid-state NMR experiments of membrane proteins because the SMA-lipid assembly both immobilizes the embedded protein and provides uniaxial order for oriented sample (OS) solid-state NMR studies. We show that aligned macrodiscs incorporating four different membrane proteins with a wide range of sizes and topological complexity yield high-resolution OS solid-state NMR spectra. The work is dedicated to Michelle Auger who made key contributions to the field of membrane and membrane protein biophysics.

Effects of deuteration on solid-state NMR spectra of single peptide crystals and oriented protein samples.

Extensive deuteration can be used to simplify NMR spectra by "diluting" and minimizing the effects of the abundant 1H nuclei. In solution-state NMR and magic angle spinning solid-state NMR of proteins, perdeuteration has been widely applied and its effects are well understood. Oriented sample solid-state NMR of proteins, however, is at a much earlier stage of development. In spite of the promise of the approach, the effects of sample deuteration are largely unknown. Here we map out the effects of perdeuteration on solid-state NMR spectra of aligned samples by closely examining differences in results obtained on fully protiated and perdeuterated samples, where all of the carbon sites have either 1H or 2H bonded to them, respectively. The 2H and 15N labeled samples are back-exchanged in 1H2O solution so that the amide 15N sites have a bonded 1H. Line-widths in the 15N chemical shift, 1H chemical shift, and 1H-15N dipolar coupling frequency dimensions were compared for peptide single crystals as well as membrane proteins aligned along with the phospholipids in bilayers with their normals perpendicular to the direction of the magnetic field. Remarkably, line-width differences were not found between fully protiated and perdeuterated samples. However, in the absence of effective 1H-1H homonuclear decoupling, the line-widths in the 1H-15N heteronuclear dipolar coupling frequency dimension were greatly narrowed in the perdeuterated samples. In proton-driven spin diffusion (PDSD) experiments, no effects of perdeuteration were observed. In contrast, in mismatched Hartmann-Hahn experiments, perdeuteration enhances cross-peak intensities by allowing more efficient spin-exchange with less polarization transfer back to the carbon-bound 1H. Here we show that in oriented sample solid-state NMR, the effects of perdeuteration can be exploited in experiments where 1H-1H homonuclear decoupling cannot be applied. These data also provide evidence for the possible contribution of direct 15N-15N dilute-spin mixing mechanism in proton-driven spin diffusion experiments.

Metal-ion Binding to Host Defense Peptide Piscidin 3 Observed in Phospholipid Bilayers by Magic Angle Spinning Solid-state NMR.

Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system. They have attracted interest as novel compounds with the potential to treat infections associated with multi-drug resistant bacteria. In this study, we investigate piscidin 3 (P3), an AMP that was first discovered in the mast cells of hybrid striped bass. Prior studies showed that P3 is less active than its homolog piscidin 1 (P1) against planktonic bacteria. However, P3 has the advantage of being less toxic to mammalian cells and more active on biofilms and persister cells. Both P1 and P3 cross bacterial membranes and co-localize with intracellular DNA but P3 is more condensing to DNA while P1 is more membrane active. Recently, we showed that both peptides coordinate Cu2+ through an amino-terminal copper and nickel (ATCUN) motif. We also demonstrated that the bactericidal effects of P3 are linked to its ability to form radicals that nick DNA in the presence of Cu2+ . Since metal binding and membrane crossing by P3 is biologically important, we apply in this study solid-state NMR spectroscopy to uniformly 13 C-15 N-labeled peptide samples to structurally characterize the ATCUN motif of P3 bound to bilayers and coordinated to Ni2+ and Cu2+ . These experiments are supplemented with density functional theory calculations. Taken together, these studies refine the arrangement of not only the backbone but also side chain atoms of an AMP simultaneously bound to metal ions and phospholipid bilayers.

Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins.

Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30 nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. The narrow resonance linewidths observed from membrane proteins in styrene-maleic acid macrodiscs advance structure determination by oriented sample solid-state NMR spectroscopy.

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues.

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW5,19ALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2H- and 15N-labeled residues. GW5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2H quadrupolar or 15N-1H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW5,19ALP23, the modified GW4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2H and 15N NMR signals confirm that the extent of dynamic averaging, particularly rotational "slippage" about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR.

The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G protein-coupled receptor, located in the cell membrane of neutrophils. Here, we describe 1H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn2+ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy.

The structure of monomeric human chemokine IL-8 (residues 1-66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1-72), with the main differences being the location of the N-loop (residues 10-22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67-72). NMR was used to analyze the interactions of monomeric IL-8 (1-66) with ND-CXCR1 (residues 1-38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1-72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.

Manipulating Protein-Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins.

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

Applications of NMR to membrane proteins.

Membrane proteins present a challenge for structural biology. In this article, we review some of the recent developments that advance the application of NMR to membrane proteins, with emphasis on structural studies in detergent-free, lipid bilayer samples that resemble the native environment. NMR spectroscopy is not only ideally suited for structure determination of membrane proteins in hydrated lipid bilayer membranes, but also highly complementary to the other principal techniques based on X-ray and electron diffraction. Recent advances in NMR instrumentation, spectroscopic methods, computational methods, and sample preparations are driving exciting new efforts in membrane protein structural biology.

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes.

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail's biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40-0.60 ppm for 13C, 0.11-0.15 ppm for 1H, and 0.46-0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH.

Structure determination of proteins by NMR is unique in its ability to measure restraints, very accurately, in environments and under conditions that closely mimic those encountered in vivo. For example, advances in solid-state NMR methods enable structure determination of membrane proteins in detergent-free lipid bilayers, and of large soluble proteins prepared by sedimentation, while parallel advances in solution NMR methods and optimization of detergent-free lipid nanodiscs are rapidly pushing the envelope of the size limit for both soluble and membrane proteins. These experimental advantages, however, are partially squandered during structure calculation, because the commonly used force fields are purely repulsive and neglect solvation, Van der Waals forces and electrostatic energy. Here we describe a new force field, and updated energy functions, for protein structure calculations with EEFx implicit solvation, electrostatics, and Van der Waals Lennard-Jones forces, in the widely used program Xplor-NIH. The new force field is based primarily on CHARMM22, facilitating calculations with a wider range of biomolecules. The new EEFx energy function has been rewritten to enable OpenMP parallelism, and optimized to enhance computation efficiency. It implements solvation, electrostatics, and Van der Waals energy terms together, thus ensuring more consistent and efficient computation of the complete nonbonded energy lists. Updates in the related python module allow detailed analysis of the interaction energies and associated parameters. The new force field and energy function work with both soluble proteins and membrane proteins, including those with cofactors or engineered tags, and are very effective in situations where there are sparse experimental restraints. Results obtained for NMR-restrained calculations with a set of five soluble proteins and five membrane proteins show that structures calculated with EEFx have significant improvements in accuracy, precision, and conformation, and that structure refinement can be obtained by short relaxation with EEFx to obtain improvements in these key metrics. These developments broaden the range of biomolecular structures that can be calculated with high fidelity from NMR restraints.

Simultaneous cross polarization to (13)C and (15)N with (1)H detection at 60kHz MAS solid-state NMR.

We describe high resolution MAS solid-state NMR experiments that utilize (1)H detection with 60kHz magic angle spinning; simultaneous cross-polarization from (1)H to (15)N and (13)C nuclei; bidirectional cross-polarization between (13)C and (15)N nuclei; detection of both amide nitrogen and aliphatic carbon (1)H; and measurement of both (13)C and (15)N chemical shifts through multi-dimensional correlation experiments. Three-dimensional experiments correlate amide (1)H and alpha (1)H selectively with (13)C or (15)N nuclei in a polypeptide chain. Two separate three-dimensional spectra correlating (1)Hα/(13)Cα/(1)H(N) and (1)H(N)/(15)N/(1)Hα are recorded simultaneously in a single experiment, demonstrating that a twofold savings in experimental time is potentially achievable. Spectral editing using bidirectional coherence transfer pathways enables simultaneous magnetization transfers between (15)N, (13)Cα(()(i)()) and (13)C'(()(i)(-1)), facilitating intra- and inter-residue correlations for sequential resonance assignment. Non-uniform sampling is integrated into the experiments, further reducing the length of experimental time.

Structural determination of virus protein U from HIV-1 by NMR in membrane environments.

Virus protein U (Vpu) from HIV-1, a small membrane protein composed of a transmembrane helical domain and two α-helices in an amphipathic cytoplasmic domain, down modulates several cellular proteins, including CD4, BST-2/CD317/tetherin, NTB-A, and CCR7. The interactions of Vpu with these proteins interfere with the immune system and enhance the release of newly synthesized virus particles. It is essential to characterize the structure and dynamics of Vpu in order to understand the mechanisms of the protein-protein interactions, and potentially to discover antiviral drugs. In this article, we describe investigations of the cytoplasmic domain of Vpu as well as full-length Vpu by NMR spectroscopy. These studies are complementary to earlier analysis of the transmembrane domain of Vpu. The results suggest that the two helices in the cytoplasmic domain form a U-shape. The length of the inter-helical loop in the cytoplasmic domain and the orientation of the third helix vary with the lipid composition, which demonstrate that the C-terminal helix is relatively flexible, providing accessibility for interaction partners.

Structure and Substrate Sequestration in the Pyoluteorin Type II Peptidyl Carrier Protein PltL.

Type II nonribosomal peptide synthetases (NRPS) generate exotic amino acid derivatives that, combined with additional pathways, form many bioactive natural products. One family of type II NRPSs produce pyrrole moieties, which commonly arise from proline oxidation while tethered to a conserved, type II peptidyl carrier protein (PCP), as exemplified by PltL in the biosynthesis of pyoluteorin. We sought to understand the structural role of pyrrole PCPs in substrate and protein interactions through the study of pyrrole analogs tethered to PltL. Solution-phase NMR structural analysis revealed key interactions in residues of helix II and III with a bound pyrrole moiety. Conservation of these residues among PCPs in other pyrrole containing pathways suggests a conserved mechanism for formation, modification, and incorporation of pyrrole moieties. Further NOE analysis provided a unique pyrrole binding motif, offering accurate substrate positioning within the cleft between helices II and III. The overall structure resembles other PCPs but contains a unique conformation for helix III. This provides evidence of sequestration by the PCP of aromatic pyrrole substrates, illustrating the importance of substrate protection and regulation in type II NRPS systems.