A Split‐Intein‐Based Method for the Efficient Production of Circularized Nanodiscs for Structural Studies of Membrane Proteins

Phospholipid nanodiscs are a native‐like membrane mimetic that is suitable for structural studies of membrane proteins. Although nanodiscs of different sizes exist for various structural applications, their thermal and long‐term stability can vary considerably. Covalently circularized nanodiscs are a perfect tool to overcome these limitations. Existing methods for the production of circularized nanodiscs can be time‐consuming and technically demanding. Therefore, an easy in vivo approach, in which circularized membrane scaffold proteins (MSPs) can be directly obtained from Escherichia coli culture, is reported herein. Nostoc punctiforme DnaE split‐intein fusions with MSPs of various lengths are used and consistently provide circularized nanodiscs in high yields. With this approach, a large variety of circularized nanodiscs, ranging from 7 to 26 nm in diameter, that are suitable for NMR spectroscopy and electron microscopy (EM) applications can be prepared. These nanodiscs are superior to those of the corresponding linear versions in terms of stability and size homogeneity, which affects the quality of NMR spectroscopy data and EM experiments. Due to their long‐term stability and homogeneity, the presented small circular nanodiscs are suited for high‐resolution NMR spectroscopy studies, as demonstrated with two membrane proteins of 17 or 32 kDa in size. The presented method will provide easy access to circularized nanodiscs for structural studies of membrane proteins and for applications in which a defined and stable nanodisc size is required.     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery

Nuclear magnetic resonance (NMR) spectroscopy is a valuable technique for ligand screening, because it exhibits high specificity toward chemical structure and interactions. Dissolution dynamic nuclear polarization (DNP) is a recent advance in NMR methodology that enables the creation of non‐equilibrium spin states, which can dramatically increase NMR sensitivity. Here, the transfer of such spin polarization from hyperpolarized ligand to protein is observed. Mixing hyperpolarized benzamidine with the serine protease trypsin, a “fingerprint” of enhanced protein signals is observed, which shows a different intensity profile than the equilibrium NMR spectrum of the protein, but coincides closely to the frequency profile of a saturation transfer difference (STD) NMR experiment. The DNP experiment benefits from hyperpolarization and enables observation of all frequencies in a single, rapid experiment. Based on these merits, it is an interesting alternative to the widely used STD experiment for identification of protein–ligand interactions.     

Characterization of a Cyclic Nucleotide‐Activated K+ Channel and its Lipid Environment by Using Solid‐State NMR Spectroscopy

Voltage‐gated ion channels are large tetrameric multidomain membrane proteins that play crucial roles in various cellular transduction pathways. Because of their large size and domain‐related mobility, structural characterization has proved challenging. We analyzed high‐resolution solid‐state NMR data on different isotope‐labeled protein constructs of a bacterial cyclic nucleotide‐activated K⁺ channel (MlCNG) in lipid bilayers. We could identify the different subdomains of the 4×355 residue protein, such as the voltage‐sensing domain and the cyclic nucleotide binding domain. Comparison to ssNMR data obtained on isotope‐labeled cell membranes suggests a tight association of negatively charged lipids to the channel. We detected spectroscopic polymorphism that extends beyond the ligand binding site, and the corresponding protein segments have been associated with mutant channel types in eukaryotic systems. These findings illustrate the potential of ssNMR for structural investigations on large membrane‐embedded proteins, even in the presence of local disorder.     

Transient Interactions of a Cytosolic Protein with Macromolecular and Vesicular Cosolutes: Unspecific and Specific Effects

Cytosolic proteins do not occur as isolated but are exposed to many interactions within a crowded cellular environment. We investigated the associations between a test cytosolic protein, human ileal bile acid binding protein (IBABP), and model cosolutes mimicking macromolecular and lipid membrane intracellular components. Using fluorescence spectroscopy, heteronuclear NMR, and molecular dynamics, we found that IBABP associated weakly with anionic lipid vesicles and experienced transient unspecific contacts with albumin. Localized dynamic perturbations were observed even in the case of apparent unspecific binding. IBABP and ubiquitin did not display mutually attractive forces, whereas IBABP associated specifically with lysozyme. A structural model of the IBABP–lysozyme complex was obtained by data‐driven docking simulation. Presumably, all the interactions shown here contribute to modulating functional communication of a protein in its native environment.     

STD‐NMR‐Based Protein Engineering of the Unique Arylpropionate‐Racemase AMDase G74C

Structure‐guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40‐fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation‐transfer‐difference NMR (STD‐NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD‐NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure‐guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD‐NMR as versatile tool for the analysis of enzyme–substrate interactions in catalytically competent systems and for the guidance of protein engineering.     

Functional Investigations of Thromboxane Synthase (CYP5A1) in Lipid Bilayers of Nanodiscs

CYP5A1 is a membrane‐associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH₂) into thromboxane A₂ (TXA₂), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER‐bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug‐metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small‐molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.     

Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding

The impact of the incorporation of a non‐natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post‐translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X‐ray crystallography. The structure and dynamics of the apo state of AZH‐modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH‐modified PDZ3 and an azulene‐linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene‐linked peptide and the isotopically labelled protein. Co‐crystallisation and soaking failed for the peptide‐bound holo complex. NMR spectroscopy, however, allowed determination of the protein–ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA‐modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target.     

Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling

G protein-coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively ¹⁹F-labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.     

Fake It ‘Till You Make It—The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics

Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular membranes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The latter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics.     

Kinase in Motion: Insights into the Dynamic Nature of p38α by High‐Pressure NMR Spectroscopic Studies

Protein kinases are highly dynamic and complex molecules. Here we present high‐pressure and relaxation studies of the activated p38α mitogen‐activated protein kinase (MAPK). p38α plays a central role in inflammatory diseases such as rheumatoid arthritis and is therefore a highly attractive pharmaceutical target. The combination of high pressure and NMR spectroscopy allowed for a detailed per‐residue based assessment of the structural plasticity of p38α and the accessibility of low‐lying excited‐energy conformations throughout the kinase structure. Such information is uniquely accessible through the combination of liquid‐state NMR and high pressure and is of considerable value for the drug discovery process. The interactions of p38α and DFG‐in and DFG‐out ligands were studied under the application of high pressure, and we demonstrate how we can alter kinase dynamics by pressure in a similar way to what has previously only been observed by ligand binding. Pressure is shown to be a mild and efficient tool for manipulation of intermediate‐timescale dynamics.     

Investigation of a KcsA Cytoplasmic pH Gate in Lipoprotein Nanodiscs

The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane–cytoplasm interface (TM2 gate), and in the cytoplasmic C-terminal domain (CTD), an amphiphilic four-helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH-induced open-to-closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl-protonated at Leu/Val residues to allow observation of both states by NMR, and spin-labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0–5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin–spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated “closed” channels. We extended these findings for isolated CTD-derived peptides to full-length KcsA and have established a contribution of the CTD to KcsA pH-controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.     

Protein Interactions with Nanoparticle Surfaces: Highlighting Solution NMR Techniques

In the last decade, nanoparticles (NPs) have become a key tool in medicine and biotechnology as drug delivery systems, biosensors and diagnostic devices. The composition and surface chemistry of NPs vary based on the materials used: typically organic polymers, inorganic materials, or lipids. Nanoparticle classes can be further divided into sub‐categories depending on the surface modification and functionalization. These surface properties matter when NPs are introduced into a physiological environment, as they will influence how nucleic acids, lipids, and proteins will interact with the NP surface. While small‐molecule interactions are easily probed using NMR spectroscopy, studying protein‐NP interactions using NMR introduces several challenges. For example, globular proteins may have a perturbed conformation when attached to a foreign surface, and the size of NP‐protein conjugates can lead to excessive line broadening. Many of these challenges have been addressed, and NMR spectroscopy is becoming a mature technique for in situ analysis of NP binding behavior. It is therefore not surprising that NMR has been applied to NP systems and has been used to study biomolecules on NP surfaces. Important considerations include corona composition, protein behavior, and ligand architecture. These features are difficult to resolve using classical surface and material characterization strategies, and NMR provides a complementary avenue of characterization. In this review, we examine how solution NMR can be combined with other analytical techniques to investigate protein behavior on NP surfaces.     

The Tyrosine Gate of the Bacterial Lectin FimH: A Conformational Analysis by NMR Spectroscopy and X‐ray Crystallography

Urinary tract infections caused by uropathogenic E. coli are among the most prevalent infectious diseases. The mannose‐specific lectin FimH mediates the adhesion of the bacteria to the urothelium, thus enabling host cell invasion and recurrent infections. An attractive alternative to antibiotic treatment is the development of FimH antagonists that mimic the physiological ligand. A large variety of candidate drugs have been developed and characterized by means of in vitro studies and animal models. Here we present the X‐ray co‐crystal structures of FimH with members of four antagonist classes. In three of these cases no structural data had previously been available. We used NMR spectroscopy to characterize FimH–antagonist interactions further by chemical shift perturbation. The analysis allowed a clear determination of the conformation of the tyrosine gate motif that is crucial for the interaction with aglycone moieties and was not obvious from X‐ray structural data alone. Finally, ITC experiments provided insight into the thermodynamics of antagonist binding. In conjunction with the structural information from X‐ray and NMR experiments the results provide a mechanism for the often‐observed enthalpy–entropy compensation of FimH antagonists that plays a role in fine‐tuning of the interaction.     

STD NMR study of the interactions between antibody 2G12 and synthetic oligomannosides that mimic selected branches of gp120 glycans

The human immunodeficiency virus type-1 (HIV-1) is able to shield immunogenic peptide epitopes on its envelope spike (a trimer of two glycoproteins, gp120 and gp41) by presenting numerous host-derived N-linked glycans. Nevertheless, broadly neutralizing antibodies against gp120 and gp41 have been isolated from HIV-1-infected patients and provide protection against viral challenge in animal models. Among these, the monoclonal antibody 2G12 binds to clusters of high-mannose-type glycans that are present on the surface of gp120. These types of glycans have thus been envisaged as target structures for the development of synthetic agents capable of eliciting 2G12-like antibodies. High-resolution structural studies of 2G12 and chemically defined glycan-type ligands, including crystallographic data, have been performed to gain an insight into this interaction. Further studies are still required to design a carbohydrate-based vaccine for HIV. Our previous NMR studies highlighted different recognition modes of two branched synthetic oligosaccharides, a penta- and a heptamannoside, by 2G12 in solution. In order to clarify the underlying structural reasons for such different behaviors, we have herein "dissected" the branches into the linear tri- and tetra- oligomannosides by chemical synthesis and studied their interactions with 2G12 in solution by saturation transfer difference (STD) NMR spectroscopy. The results confirm the distinct preferences of 2G12 for the studied branches and afford explanations for the observed differences. This study provides important structural information for further ligand optimizations. Possible effects of structural modifications on the solvent-exposed end of the ligands are also discussed.     

Evidence for Novel Action at the Cell‐Binding Site of Human Angiogenin Revealed by Heteronuclear NMR Spectroscopy,in silico and in vivo Studies

A member of the ribonuclease A superfamily, human angiogenin (hAng) is a potent angiogenic factor. Heteronuclear NMR spectroscopy combined with induced‐fit docking revealed a dual binding mode for the most antiangiogenic compound of a series of ribofuranosyl pyrimidine nucleosides that strongly inhibit hAng's angiogenic activity in vivo. While modeling suggests the potential for simultaneous binding of the inhibitors at the active and cell‐binding sites, NMR studies indicate greater affinity for the cell‐binding site than for the active site. Additionally, molecular dynamics simulations at 100 ns confirmed the stability of binding at the cell‐binding site with the predicted protein–ligand interactions, in excellent agreement with the NMR data. This is the first time that a nucleoside inhibitor is reported to completely inhibit the angiogenic activity of hAng in vivo by exerting dual inhibitory activity on hAng, blocking both the entrance of hAng into the cell and its ribonucleolytic activity.     

Structure and Stability of Carbohydrate–Lipid Interactions. Methylmannose Polysaccharide–Fatty Acid Complexes

We report a detailed study of the structure and stability of carbohydrate–lipid interactions. Complexes of a methylmannose polysaccharide (MMP) derivative and fatty acids (FAs) served as model systems. The dependence of solution affinities and gas‐phase dissociation activation energies (E_a) on FA length indicates a dominant role of carbohydrate–lipid interactions in stabilizing (MMP+FA) complexes. Solution ¹H NMR results reveal weak interactions between MMP methyl groups and FA acyl chain; MD simulations suggest the complexes are disordered. The contribution of FA methylene groups to the E_a is similar to that of heats of transfer of n‐alkanes from the gas phase to polar solvents, thus suggesting that MMP binds lipids through dipole‐induced dipole interactions. The MD results point to hydrophobic interactions and H‐bonds with the FA carboxyl group. Comparison of collision cross sections of deprotonated (MMP+FA) ions with MD structures suggests that the gaseous complexes are disordered.     

STD‐NMR Studies Suggest that Two Acceptor Substrates for GlfT2, a Bifunctional Galactofuranosyltransferase Required for the Biosynthesis of Mycobacterium tuberculosis Arabinogalactan,Compete for the Same Binding Site

The mycobacterial cell wall is a complex architecture, which has, as its major structural component, a lipidated polysaccharide covalently bound to peptidoglycan. This structure, termed the mycolyl–arabinogalactan–peptidoglycan complex, possesses a core galactan moiety composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating β‐(1→6) and β‐(1→5) linkages. Recent studies have shown that the entire galactan is synthesized by the action of only two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation‐transfer difference (STD) NMR spectroscopy studies with GlfT2 using two trisaccharide acceptor substrates, β‐D ‐Galf‐(1→6)‐β‐D ‐Galf‐(1→5)‐β‐D ‐Galf‐O(CH₂)₇CH₃ ( 2 ) and β‐D ‐Galf‐(1→5)‐β‐D ‐Galf‐(1→6)‐β‐D ‐Galf‐O(CH₂)₇CH₃ ( 3 ), as well as the donor substrate for the enzyme, UDP‐Galf. Competition STD‐NMR titration experiments and saturation transfer double difference (STDD) experiments with 2 and 3 were undertaken to explore the bifunctionality of this enzyme, in particular to answer whether one or two active sites are responsible for the formation of both β‐(1→5)‐ and β‐(1→6)‐Galf linkages. It was demonstrated that 2 and 3 bind competitively at the same site; this suggests that GlfT2 has one active site pocket capable of catalyzing both β‐(1→5) and β‐(1→6) galactofuranosyl transfer reactions. The addition of UDP‐Galf to GlfT2 in the presence of either 2 or 3 generated a tetrasaccharide product; this indicates that the enzyme was catalytically active under the conditions at which the STD‐NMR experiments were carried out.     

Discovery of New E‐Selectin Inhibitors by Virtual Screening,Fluorescence Binding Assays,and STD NMR Experiments

E‐selectin is an endothelial protein that participates in the adhesion of metastatic cancer cells, and is therefore a relevant target for antitumor therapeutic intervention. In this work, virtual screening was used to identify new E‐selectin inhibitors from a subset of drug‐like molecules retrieved from the ZINC database, including the physiological ligand sLe^x as reference structure (PDB ID: 1G1T ). Four hits were chosen and subjected to molecular dynamics simulations and fluorescence binding assays, which led to the determination of experimental dissociation constants between 333 and 1012 μm . The candidate with the highest affinity was studied by saturation transfer difference (STD) NMR experiments and complete relaxation and conformational exchange matrix analysis of saturation transfer (CORCEMA‐ST), aimed at identifying the preferable binding mode with E‐selectin. Our results revealed that this new inhibitor binds more strongly than sLe^x in the E‐selectin binding site, in good agreement with simulation predictions. These properties will prove valuable for the future design of drugs that target E‐selectin.