Longitudinal and Transverse 1H Nuclear Magnetic Resonance Relaxivities of Lanthanide Ions in Aqueous Solution up to 1.4 GHz/33 T.

The longitudinal and transverse nuclear magnetic resonance relaxivity dispersion (NMRD) of 1H in water induced by the paramagnetic relaxation enhancement (PRE) of dissolved lanthanide ions (Ln3+) can become very strong. Longitudinal and transverse 1H NMRD for Gd3+, Dy3+, Er3+ and Ho3+ were measured from 20 MHz/0.47 T to 1382 MHz/32.5 T, which extended previous studies by a factor of more than two in the frequency range. For the NMRD above 800 MHz, we used a resistive magnet, which exhibits reduced field homogeneity and stability in comparison to superconducting and permanent NMR magnets. These drawbacks were addressed by dedicated NMRD methods. In a comparison of NMRD measurements between 800 MHz and 950 MHz performed in both superconducting and resistive magnets, it was found that the longitudinal relaxivities were almost identical. However, the magnetic field fluctuations of the resistive magnet strongly perturbed the transverse relaxation. The longitudinal NMRDs are consistent with previous work up to 600 MHz. The transverse NMRD nearly scales with the longitudinal one with a factor close to one. The data can be interpreted within a PRE model that comprises the dipolar hyperfine interactions between the 1H and the paramagnetic ions, as well as a Curie spin contribution that is dominant at high magnetic fields for Dy3+, Er3+ and Ho3+. Our findings provide a solid methodological basis and valuable quantitative insights for future high-frequency NMRD studies, enhancing the measurement accuracy and applicability of PRE models for paramagnetic ions in aqueous solutions.

Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach: application to anti-LAMP1 Fab.

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13C', 13Cα, 13Cß resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs' higher order structures, dynamics and interactions, using solution-state NMR.

Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties.

Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single-particle-tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side-chain protonation of the conserved residues E212 and H62,  altering the hydrogen-bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green-to-Red photoconversion properties differ between the A and B states. The chromophore in the A-state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B-state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.

Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface.

RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.

The plasma membrane-associated cation-binding protein PCaP1 of Arabidopsis thaliana is a uranyl-binding protein.

Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.

Visualizing the transiently populated closed-state of human HSP90 ATP binding domain.

HSP90 are abundant molecular chaperones, assisting the folding of several hundred client proteins, including substrates involved in tumor growth or neurodegenerative diseases. A complex set of large ATP-driven structural changes occurs during HSP90 functional cycle. However, the existence of such structural rearrangements in apo HSP90 has remained unclear. Here, we identify a metastable excited state in the isolated human HSP90α ATP binding domain. We use solution NMR and mutagenesis to characterize structures of both ground and excited states. We demonstrate that in solution the HSP90α ATP binding domain transiently samples a functionally relevant ATP-lid closed state, distant by more than 30 Å from the ground state. NMR relaxation enables to derive information on the kinetics and thermodynamics of this interconversion, while molecular dynamics simulations establish that the ATP-lid in closed conformation is a metastable exited state. The precise description of the dynamics and structures sampled by human HSP90α ATP binding domain provides information for the future design of new therapeutic ligands.

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.

The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features.

In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.

Assembly of The Mitochondrial Complex†I Assembly Complex Suggests a Regulatory Role for Deflavination.

Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.

Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis.

The initial greening of angiosperms involves light activation of photoreceptors that trigger photomorphogenesis, followed by the development of chloroplasts. In these semi-autonomous organelles, construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here, we show that the expression of PAP8, an essential subunit of the plastid-encoded RNA polymerase (PEP) in Arabidopsis thaliana, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 protein is localized and active in both plastids and the nucleus, and particularly required for the formation of late photobodies. In the pap8 albino mutant, phytochrome-mediated signalling is altered, degradation of the chloroplast development repressors PIF1/PIF3 is disrupted, HY5 is not stabilized, and the expression of the photomorphogenesis regulator GLK1 is impaired. PAP8 translocates into plastids via its targeting pre-sequence, interacts with the PEP and eventually reaches the nucleus, where it can interact with another PEP subunit pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome B-mediated signalling cascade and the reshaping of the PEP activity, it may coordinate nuclear gene expression with PEP-driven chloroplastic gene expression during chloroplast biogenesis.

Exolysin (ExlA) from Pseudomonas aeruginosa Punctures Holes into Target Membranes Using a Molten Globule Domain.

Bacteria employ several mechanisms, and most notably secretion systems, to translocate effectors from the cytoplasm to the extracellular environment or the cell surface. Pseudomonas aeruginosa widely employs secretion machineries such as the Type III Secretion System to support virulence and cytotoxicity. However, recently identified P. aeruginosa strains that do not express the Type III Secretion System have been shown to express ExlA, an exolysin translocated through a two-partner secretion system, and are the causative agents of severe lung hemorrhage. Sequence predictions of ExlA indicate filamentous hemagglutinin (FHA-2) domains as the prevalent features, followed by a C-terminal domain with no known homologs. In this work, we have addressed the mechanism employed by ExlA to target membrane bilayers by using NMR, small-angle X-ray scattering, atomic force microscopy, and cellular infection techniques. We show that the C-terminal domain of ExlA displays a "molten globule-like" fold that punctures small holes into membranes composed of negatively charged lipids, while other domains could play a lesser role in target recognition. In addition, epithelial cells infected with P. aeruginosa strains expressing different ExlA variants allow localization of the toxin to lipid rafts. ExlA homologs have been identified in numerous bacterial strains, indicating that lipid bilayer destruction is an effective strategy employed by bacteria to establish interactions with multiple hosts.

ssNMRlib: a comprehensive library and tool box for acquisition of solid-state nuclear magnetic resonance experiments on Bruker spectrometers.

We introduce ssNMRlib, a comprehensive suite of pulse sequences and jython scripts for user-friendly solid-state nuclear magnetic resonance (NMR) data acquisition, parameter optimization and storage on Bruker spectrometers. ssNMRlib allows the straightforward setup of even highly complex multi-dimensional solid-state NMR experiments with a few clicks from an intuitive graphical interface directly from the Bruker Topspin acquisition software. ssNMRlib allows the setup of experiments in a magnetic-field-independent manner and thus facilitates the workflow in a multi-spectrometer setting with a centralized library. Safety checks furthermore assist the user in experiment setup. Currently hosting more than 140 1D to 4D experiments, primarily for biomolecular solid-state NMR, the library can be easily customized and new experiments are readily added as new templates. ssNMRlib is part of the previously introduced NMRlib library, which comprises many solution-NMR pulse sequences and macros.

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex.

Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.

NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers.

We present NMRlib, a suite of jython-based tools designed for Bruker spectrometers (TopSpin versions 3.2-4.0) that allow easy setup, management, and exchange of NMR experiments. A NMR experiment can be set up and executed in a few clicks by navigating through the NMRlib GUI tree structure, without any further parameter adjustment. NMRlib is magnetic-field independent, and thus particularly helpful for laboratories operating multiple NMR spectrometers. NMRlib is easily personalized by adding, deleting, or reorganizing experiments. Additional tools are provided for data processing, visualization, and analysis. In particular, NMRlib contains all the polarization-enhanced fast-pulsing NMR experiments (SOFAST, BEST, HADAMAC,…) developed in our laboratory over the last decade. We also discuss some specific features that have been implemented to make these experiments most efficient and user friendly.

Aromatic SOFAST-HMBC for proteins at natural 13C abundance.

We propose here SOFAST-HMBC as a new complementary NMR tool for aromatic side chain assignment of protein samples at natural 13C abundance. The characteristic peak patterns detected in SOFAST-HMBC for each aromatic side chain allow straightforward assignment of all protons and carbons (including quaternary ones) of the aromatic ring, and for tyrosine and phenylalanine, connection to the CB of the aliphatic chain. The performance of SOFAST-HMBC is demonstrated for three small proteins (7-14 kDa) at millimolar sample concentration using modern high-field NMR instruments equipped with cryogenically cooled probes. Despite the low amount of NMR-active 13C nuclei in these samples, 1H-13C multiple-bond correlation spectra of good quality were obtained in reasonable experimental times of typically less than 24 h.

Optimized fast mixing device for real-time NMR applications.

We present an improved fast mixing device based on the rapid mixing of two solutions inside the NMR probe, as originally proposed by Hore and coworkers (J. Am. Chem. Soc. 125 (2003) 12484-12492). Such a device is important for off-equilibrium studies of molecular kinetics by multidimensional real-time NMR spectrsocopy. The novelty of this device is that it allows removing the injector from the NMR detection volume after mixing, and thus provides good magnetic field homogeneity independently of the initial sample volume placed in the NMR probe. The apparatus is simple to build, inexpensive, and can be used without any hardware modification on any type of liquid-state NMR spectrometer. We demonstrate the performance of our fast mixing device in terms of improved magnetic field homogeneity, and show an application to the study of protein folding and the structural characterization of transiently populated folding intermediates.

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.

Probing Conformational Exchange Dynamics in a Short-Lived Protein Folding Intermediate by Real-Time Relaxation-Dispersion NMR.

NMR spectroscopy is a powerful tool for studying molecular dynamics at atomic resolution simultaneously for a large number of nuclear sites. In this communication, we combine two powerful NMR techniques, relaxation-dispersion NMR and real-time NMR, in order to obtain unprecedented information on the conformational exchange dynamics present in short-lived excited protein states, such as those transiently accumulated during protein folding. We demonstrate the feasibility of the approach for the amyloidogenic protein ß2-microglobulin that folds via an intermediate state which is believed to be responsible for the onset of the aggregation process leading to amyloid formation.

Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE.

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5'-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.

Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.

Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.