Simultaneous use of solution, solid-state NMR and X-ray crystallography to study the conformational landscape of the Crh protein during oligomerization and crystallization

We explore, using the Crh protein dimer as a model, how information from solution NMR, solid-state NMR and X-ray crystallography can be combined using structural bioinformatics methods, in order to get insights into the transition from solution to crystal. Using solid-state NMR chemical shifts, we filtered intra-monomer NMR distance restraints in order to keep only the restraints valid in the solid state. These filtered restraints were added to solid-state NMR restraints recorded on the dimer state to sample the conformational landscape explored during the oligomerization process. The use of non-crystallographic symmetries then permitted the extraction of converged conformers subsets. Ensembles of NMR and crystallographic conformers calculated independently display similar variability in monomer orientation, which supports a funnel shape for the conformational space explored during the solution-crystal transition. Insights into alternative conformations possibly sampled during oligomerization were obtained by analyzing the relative orientation of the two monomers, according to the restraint precision. Molecular dynamics simulations of Crh confirmed the tendencies observed in NMR conformers, as a paradoxical increase of the distance between the two β1a strands, when the structure gets closer to the crystallographic structure, and the role of water bridges in this context

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

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

International audienceWe 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 13^{13}C abundance

International audienceWe propose here SOFAST-HMBC as a new complementary NMR tool for aromatic side chain assignment of protein samples at natural $^{13}$C 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 $^{13}$C nuclei in these samples, $^1$H-$^{13}$C multiple-bond correlation spectra of good quality were obtained in reasonable experimental times of typically less than 24 h

HNCA+, HNCO+, and HNCACB+ experiments: improved performance by simultaneous detection of orthogonal coherence transfer pathways

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Optimized fast mixing device for real-time NMR applications.

International audienceWe 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

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

International audienceNMR 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 β2-microglobulin that folds via an intermediate state which is believed to be responsible for the onset of the aggregation process leading to amyloid formation

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 shortlived excited protein states, such as those transiently accumulated during protein folding. We demonstrate the feasibility of the approach for the amyloidogenic protein β2-microglobulin that folds via an intermediate state which is believed to be responsible for the onset of the aggregation process leading to amyloid formation.This work used the NMR platform of the Grenoble Instruct Centre (ISBG: UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB)

New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.

Zinc (Zn(2+)) homeostasis is critical for pathogen host colonization and invasion. Polyhistidine triad (Pht) proteins, located at the surface of various streptococci, have been proposed to be involved in Zn(2+) homeostasis. The phtD gene, coding for a Zn(2+)-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn(2+) transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approaches. Immuno-precipitation experiments on purified membranes of Streptococcus pneumoniae (S. pneumoniae) demonstrate that native PhtD and AdcAII interact in vivo confirming our previous in vitro observations. NMR was used to demonstrate Zn(2+) transfer from the Zn(2+)-bound form of a 137 amino acid N-terminal domain of PhtD (t-PhtD) to AdcAII. The high resolution NMR structure of t-PhtD shows that Zn(2+) is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63. Comparison of the NMR parameters measured for apo- and Zn(2+)-t-PhtD shows that the loss of Zn(2+) leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume. Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn(2+) scavenger for later release to the surface transporter AdcAII, leading to Zn(2+) uptake

CopK from Cupriavidus metallidurans CH34 Binds Cu(I) in a Tetrathioether Site: Characterization by X-ray Absorption and NMR Spectroscopy

International audienceCupriavidus metallidurans CH34 is a bacterium that is resistant to high metal concentrations in the environment. Increased copper resistance is associated with the cop cluster on the large plasmid pMOL30 that is composed of at least 21 genes. The copK gene encodes a 74 residue periplasmic protein whose expression is strongly upregulated in the presence of copper. CopK was previously shown to cooperatively bind Cu(I) and Cu(II) in distinct, specific sites. The solution structure of Cu(I)−CopK and the characterization of the Cu(I) site by X-ray absorption spectroscopy and NMR are reported here. EXAFS spectra are in agreement with a tetrathioether Cu(I) site, providing so far unique spectral information on a 4S-coordinated Cu(I) in a protein. The methionine residues forming the Cu(I) site, M28, M38, M44, and M54, are identified by NMR. We propose the chemical shift of the methionine Cε as a new and sensitive probe for the detection of Cu(I) bound to thioether groups. The solution structure of Cu(I)−CopK demonstrates that Cu(I) binding induces a complete structural modification with the disruption of the second β-sheet and a rotation of the C-terminal part of nearly 180° around a hinge formed by asparagine 57. This conformational change is directly related to the loss of the dimer interface and most probably to the formation of the Cu(II) site involving histidine 70. The solution structure of Cu(I)−CopK therefore provides the molecular basis for the understanding of the Cu(I)/Cu(II) binding cooperativity