Crosstalk between regulatory elements in disordered TRPV4 N-terminus modulates lipid-dependent channel activity.

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and the network of antagonistic regulatory elements it encodes. These modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.

Synthesis of Complex Thiazoline-Containing Peptides by Cyclodesulfhydration of N-Thioacyl-2-Mercaptoethylamine Derivatives.

Herein we report a mild, efficient, and epimerization-free method for the synthesis of peptide-derived 2-thiazolines and 5,6-dihydro-4H-1,3-thiazines based on a cyclodesulfhydration of N-thioacyl-2-mercaptoethylamine or N-thioacyl-3-mercaptopropylamine derivatives. The described reaction can be easily carried out in aqueous solutions at room temperature and it is triggered by change of the pH, leading to complex thiazoline or dihydrothiazine derivatives without epimerization in excellent to quantitative yields. The new method was applied in the total synthesis of the marine metabolite mollamide F, resulting in the revision of its stereochemistry.

Extent of intrinsic disorder and NMR chemical shift assignments of the distal N-termini from human TRPV1, TRPV2 and TRPV3 ion channels.

The mammalian Transient Receptor Potential Vanilloid (TRPV) channels are a family of six tetrameric ion channels localized at the plasma membrane. The group I members of the family, TRPV1 through TRPV4, are heat-activated and exhibit remarkable polymodality. The distal N-termini of group I TRPV channels contain large intrinsically disordered regions (IDRs), ranging from ~ 75 amino acids (TRPV2) to ~ 150 amino acids (TRPV4), the vast majority of which is invisible in the structural models published so far. These IDRs provide important binding sites for cytosolic partners, and their deletion is detrimental to channel activity and regulation. Recently, we reported the NMR backbone assignments of the distal TRPV4 N-terminus and noticed some discrepancies between the extent of disorder predicted solely based on protein sequence and from experimentally determined chemical shifts. Thus, for an analysis of the extent of disorder in the distal N-termini of all group I TRPV channels, we now report the NMR assignments for the human TRPV1, TRPV2 and TRPV3 IDRs.

Backbone and side chain resonance assignment of the intrinsically disordered human DBNDD1 protein.

The dysbindin domain-containing protein 1 (DBNDD1) is a conserved protein among higher eukaryotes whose structure and function are poorly investigated so far. Here, we present the backbone and side chain nuclear magnetic resonance assignments for the human DBNDD1 protein. Our chemical-shift based secondary structure analysis reveals the human DBNDD1 as an intrinsically disordered protein.

NMR of intrinsically disordered proteins: A note on the application of 15N-13Cα het-TOCSY mixing for 13Cα magnetisation transfers.

Intrinsically disordered proteins (IDPs) or protein regions represent functionally important biomolecules without unique structure. Their inherent flexibility prevents high-resolution structure determination by X-ray or cryo-EM methods. In contrast, NMR spectroscopy provides an extensive and still growing set of experimental approaches to obtain detailed information on structure and dynamics of IDPs. Here, it is experimentally demonstrated that 15N-13Cα band-selective heteronuclear cross-polarisation that has been successfully employed recently to achieve the efficient transfer of 15Nx magnetisation from amino acid residue 'i' to 'i + 1' and 'i - 1' residues in uniformly (15N,13C)-labelled intrinsically disordered proteins can also be applied to transfer, without significant relaxation losses, 13Cαx magnetisation from an amino acid residue to its neighbouring residues. The possibility to obtain in one-shot correlation spectra arising from the simultaneous transfer of 15Nx and 13Cαx magnetisations from an amino acid residue to neighbouring residues is also demonstrated.

Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state.

ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

Fragment screening using biolayer interferometry reveals ligands targeting the SHP-motif binding site of the AAA+ ATPase p97.

Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. The AAA+ ATPase p97 is an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors address its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. Here, we describe a biolayer interferometry-based fragment screen targeting the N-domain of p97 and demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97.

Backbone and nearly complete side-chain chemical shift assignments reveal the human uncharacterized protein CXorf51A as intrinsically disordered.

Even though the human genome project showed that our DNA contains a mere 20,000 to 25,000 protein coding genes, an unexpectedly large number of these proteins remain functionally uncharacterized. A structural characterization of these "unknown" proteins may help to identify possible cellular tasks. We therefore used a combination of bioinformatics and nuclear magnetic resonance spectroscopy to structurally de-orphanize one of these gene products, the 108 amino acid human uncharacterized protein CXorf51A. Both our bioinformatics analysis as well as the [Formula: see text]H, [Formula: see text]C, [Formula: see text]N backbone and near-complete side-chain chemical shift assignments indicate that it is an intrinsically disordered protein.

15N NMR studies provide insights into physico-chemical properties of room-temperature ionic liquids.

Ionic liquids (ILs) have gained a lot of attention as alternative solvents in many fields of science in the last two decades. It is known that the type of anion has a significant influence on the macroscopic properties of the IL. To gain insights into the molecular mechanisms responsible for these effects it is important to characterize these systems at the microscopic level. Such information can be obtained from nuclear spin-relaxation studies which for compounds with natural isotope abundance are typically performed using direct 1H or 13C measurements. Here we used direct 15N measurements to characterize spin relaxation of non-protonated nitrogens in imidazolium-based ILs which are liquid at ambient temperature. We report heteronuclear 1H-15N scalar coupling constants (nJHN) and 15N relaxation parameters for non-protonated nitrogens in ten 1-ethyl-3-methylimidazolium ([C2C1IM]+)-based ILs containing a broad range of anions. The 15N relaxation rates and steady-state heteronuclear 15N-{1H} NOEs were measured using direct 15N detection at 293.2 K and two magnetic field strengths, 9.4 T and 16.4 T. The experimental data were analyzed to determine hydrodynamic characteristics of ILs and to assess the contributions to 15N relaxation from 15N chemical shift anisotropy and from 1H-15N dipolar interactions with non-bonded protons. We found that the rotational correlation times of the [C2C1IM]+ cation determined from 15N relaxation measurements at room temperature correlate linearly with the macroscopic viscosity of the ILs. Depending on the selected anion, the 15N relaxation characteristics of [C2C1IM]+ differ considerably reflecting the influence of the anion on the physicochemical properties of the IL.

Backbone and nearly complete side-chain chemical shift assignments of the human death-associated protein 1 (DAP1).

Death-associated protein 1 (DAP1) is a proline-rich cytoplasmatic protein highly conserved in most eukaryotes. It has been reported to be involved in controlling cell growth and migration, autophagy and apoptosis. The presence of human DAP1 is associated to a favourable prognosis in different types of cancer. Here we describe the almost complete [Formula: see text], [Formula: see text], and [Formula: see text] chemical shift assignments of the human DAP1. The limited spectral dispersion, mainly in the [Formula: see text] region, and the lack of defined secondary structure elements, predicted based on chemical shifts, identifies human DAP1 as an intrinsically disordered protein (IDP). This work lays the foundation for further structural investigations, dynamic studies, mapping of potential interaction partners or drug screening and development.

Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR.

Disulfide bridges establish a fundamental element in the molecular architecture of proteins and peptides which are involved e.g., in basic biological processes or acting as toxins. NMR spectroscopy is one method to characterize the structure of bioactive compounds including cystine-containing molecules. Although the disulfide bridge itself is invisible in NMR, constraints obtained via the neighboring NMR-active nuclei allow to define the underlying conformation and thereby to resolve their functional background. In this mini-review we present shortly the impact of cysteine and disulfide bonds in the proteasome from different domains of life and give a condensed overview of recent NMR applications for the characterization of disulfide-bond containing biomolecules including advantages and limitations of the different approaches.

1H, 13C, and 15N Backbone assignments of the human brain and acute leukemia cytoplasmic (BAALC) protein.

The brain and acute leukemia cytoplasmic (BAALC; UniProt entry Q8WXS3) is a 180-residue-long human protein having six known isoforms. BAALC is expressed in either hematopoietic or neuroectodermal cells and its specific function is still to be revealed. However, as a presumably membrane-anchored protein at the cytoplasmic side it is speculated that BAALC exerts its function at the postsynaptic densities of certain neurons and might play a role in developing cytogenetically normal acute myeloid leukemia (CN-AML) when it is highly overexpressed by myeloid or lymphoid progenitor cells. In order to better understand the physiological role of BAALC and to provide the basis for a further molecular characterization of BAALC, we report here the 1H, 13C, and 15N resonance assignments for the backbone nuclei of its longest hematopoietic isoform (isoform 1). In addition, we present a 1HN and 15NH chemical shift comparison of BAALC with its shortest, neuroectodermal isoform (isoform 6) which shows only minor changes in the 1H and 15N chemical shifts.

NMR experiments on the transient interaction of the intrinsically disordered N-terminal peptide of cystathionine-ß-synthase with heme.

The N-terminal segment of human cystathionine-ß-synthase (CBS(1-40)) constitutes an intrinsically disordered protein stretch that transiently interacts with heme. We illustrate that the HCBCACON experimental protocol provides an efficient alternative approach for probing transient interactions of intrinsically disordered proteins with heme in situations where the applicability of the conventional [1H, 15N]-HSQC experiment may be limited. This experiment starting with the excitation of protein side chain protons delivers information about the proline residues and thereby makes it possible to use these residues in interaction mapping experiments. Employing this approach in conjunction with site-specific mutation we show that transient heme binding is mediated by the Cys15-Pro16 motif of CBS(1-40).

1H, 13C, and 15N resonance assignments of the cytokine interleukin-36ß isoform-2.

Interleukins are cytokines performing central tasks in the human immune system. Interleukin-36ß (IL-36ß) is a member of the interleukin-1 superfamily as are its homologues IL-36α and IL-36γ. All of them interact with a common receptor composed of IL-36R and IL-1R/acP. IL-36 cytokines can activate IL-36R to proliferation of CD4 + lymphocytes or stimulate M2 macrophages as potently as IL-1ß. Within our efforts to study the structure-function relationship of the three interleukins IL-36α, IL-36ß and IL-36γ by heteronuclear multidimensional NMR, we here report the 1H, 13C, and 15N resonance assignments for the backbone and side chain nuclei of cytokine interleukin-36ß isoform-2.

Reorientation dynamics and ion diffusivity of neat dimethylimidazolium dimethylphosphate probed by NMR spectroscopy.

NMR spectroscopy at two magnetic field strengths was employed to investigate the dynamics of dimethylimidazolium dimethylphosphate ([C1C1IM][(CH3)2PO4]). [C1C1IM][(CH3)2PO4] is a low-melting, halogen-free ionic liquid comprising of only methyl groups. 13C spin-lattice relaxation rates as well as self-diffusion coefficients were measured for [C1C1IM][(CH3)2PO4] as a function of temperature. The rotational correlation times, τ c, for the cation and the anion were obtained from the 13C spin-lattice relaxation rates. Although from a theoretical point of view cations and anions are similar in size, they show different reorientation mobilities and diffusivities. The self-diffusion coefficients and the rotational correlation times were related to the radii of the diffusing spheres. The analysis reveals that the radii of the cation and the anion, respectively, are different from each other but constant at temperatures ranging from 293 to 353 K. The experimental results are rationalised by a discrete and individual cation and anion diffusion. The [(CH3)2PO4]- anion reorients faster compared to the cation but diffuses significantly slower indicating the formation of anionic aggregates. Relaxation data were acquired with standard liquid and magic-angle-spinning NMR probes to estimate residual dipolar interactions, chemical shift anisotropy or differences in magnetic susceptibility within the sample.

NMR spectroscopic studies of a TAT-derived model peptide in imidazolium-based ILs: influence on chemical shifts and the cis/trans equilibrium state.

NMR spectroscopy was used to study systematically the impact of imidazolium-based ionic liquid (IL) solutions on a TAT-derived model peptide containing Xaa-Pro peptide bonds. The selected IL anions cover a wide range of the Hofmeister series of ions. Based on highly resolved one- and two-dimensional NMR spectra individual 1H and 13C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived. The observed chemical shift changes indicate significant interactions between the peptide and the ILs. In addition, we examined the impact of different ILs towards the cis/trans equilibrium state of the Xaa-Pro peptide bonds. In this context, the IL cations appear to be of exceptional importance for inducing an alteration of the native cis/trans equilibrium state of Xaa-Pro bonds in favour of the trans-isomers.

Characterization of the interaction between the small RNA-encoded peptide SR1P and GapA from Bacillus subtilis.

Small regulatory RNAs (sRNAs) are the most prominent post-transcriptional regulators in all kingdoms of life. A few of them, e.g. SR1 from Bacillus subtilis, are dual-function sRNAs. SR1 acts as a base-pairing sRNA in arginine catabolism and as an mRNA encoding the small peptide SR1P in RNA degradation. Both functions of SR1 are highly conserved among 23 species of Bacillales. Here, we investigate the interaction between SR1P and GapA by a combination of in vivo and in vitro methods. De novo prediction of the structure of SR1P yielded five models, one of which was consistent with experimental circular dichroism spectroscopy data of a purified, synthetic peptide. Based on this model structure and a comparison between the 23 SR1P homologues, a series of SR1P mutants was constructed and analysed by Northern blotting and co-elution experiments. The known crystal structure of Geobacillus stearothermophilus GapA was used to model SR1P onto this structure. The hypothetical SR1P binding pocket, composed of two α-helices at both termini of GapA, was investigated by constructing and assaying a number of GapA mutants in the presence and absence of wild-type or mutated SR1P. Almost all residues of SR1P located in the two highly conserved motifs are implicated in the interaction with GapA. A critical lysine residue (K332) in the C-terminal α-helix 14 of GapA corroborated the predicted binding pocket.

(1)H, (13)C, and (15)N resonance assignments for the pro-inflammatory cytokine interleukin-36α.

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the (1)H, (13)C, and (15)N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.

A Set of Efficient nD NMR Protocols for Resonance Assignments of Intrinsically Disordered Proteins.

The RF pulse scheme RN[N-CA HEHAHA]NH, which provides a convenient approach to the acquisition of different multidimensional chemical shift correlation NMR spectra leading to backbone resonance assignments, including those of the proline residues of intrinsically disordered proteins (IDPs), is experimentally demonstrated. Depending on the type of correlation data required, the method involves the generation of in-phase ((15) N)(x) magnetisation via different magnetisation transfer pathways such as H→N→CO→N, HA→CA→CO→N, H→N→CA→N and H→CA→N, the subsequent application of (15) N-(13) C(α) heteronuclear Hartmann-Hahn mixing over a period of ≈100 ms, chemical-shift labelling of relevant nuclei before and after the heteronuclear mixing step and amide proton detection in the acquisition dimension. It makes use of the favourable relaxation properties of IDPs and the presence of (1) JCαN and (2) JCαN couplings to achieve efficient correlation of the backbone resonances of each amino acid residue "i" with the backbone amide resonances of residues "i-1" and "i+1". It can be implemented in a straightforward way through simple modifications of the RF pulse schemes commonly employed in protein NMR studies. The efficacy of the approach is demonstrated using a uniformly ((15) N,(13) C) labelled sample of α-synuclein. The different possibilities for obtaining the amino-acid-type information, simultaneously with the connectivity data between the backbone resonances of sequentially neighbouring residues, have also been outlined.

Solvent Removal Induces a Reversible ß-to-α Switch in Oligomeric Aß Peptide.

Solvation and hydration are key factors for determining the stability and folding of proteins, as well as the formation of amyloid fibrils and related polypeptide aggregates. Using attenuated total reflectance Fourier-transform infrared and solid-state NMR spectroscopy, we find that the Aß peptide experiences a remarkable conformational switch from ß to α secondary structure upon solvent removal by lyophilization of oligomers. This transition is, contrary to Aß fibrils, independent of concentration of organic co-solvents or co-solutes and is reversible upon re-addition of the solvent. Our data illuminate a previously unnoted secondary structural plasticity of the Aß peptide in amyloid oligomers that could bear relevance for Aß's interactions with cellular structures of low polarity.