Antoine Loquet

Atomic model of the type III secretion system needle

Pathogenic bacteria using a type III secretion system (T3SS) to manipulate host cells cause many different infections including Shigella dysentery, typhoid fever, enterohaemorrhagic colitis and bubonic plague. An essential part of the T3SS is a hollow needle-like protein filament through which effector proteins are injected into eukaryotic host cells. Currently, the three-dimensional structure of the needle is unknown because it is not amenable to X-ray crystallography and solution NMR, as a result of its inherent non-crystallinity and insolubility. Cryo-electron microscopy combined with crystal or solution NMR subunit structures has recently provided a powerful hybrid approach for studying supramolecular assemblies, resulting in low-resolution and medium-resolution models. However, such approaches cannot deliver atomic details, especially of the crucial subunit–subunit interfaces, because of the limited cryo-electron microscopic resolution obtained in these studies. Here we report an alternative approach combining recombinant wild-type needle production, solid-state NMR, electron microscopy and Rosetta modelling to reveal the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. We show that the 80-residue subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellar filament of S. typhimurium. In contrast to established models of the needle in which the amino terminus of the protein subunit was assumed to be α-helical and positioned inside the needle, our model reveals an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen.

Atomic-Resolution Three-Dimensional Structure of HET-s(218-289) Amyloid Fibrils by Solid-State NMR Spectroscopy

We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the prion domain of the fungal prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218-289) in its fibrillar state forms a left-handed β-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.

Characterization of different water pools in solid-state NMR protein samples

We observed and characterized two distinct signals originating from different pools of water protons in solid-state NMR protein samples, namely from crystal water which exchanges polarization with the protein (on the NMR timescale) and is located in the protein-rich fraction at the periphery of the magic-angle spinning (MAS) sample container, and supernatant water located close to the axis of the sample container. The polarization transfer between the water and the protein can be probed by two-dimensional exchange spectroscopy, and we show that the supernatant water does not interact with protein on the timescale of the experiments. The two water pools have different spectroscopic properties, including resonance frequency, longitudinal, transverse and rotating frame relaxation times. The supernatant water can be removed almost completely physically or can be frozen selectively. Both measures lead to an enhancement of the quality factor of the probe circuit, accompanied by an improvement of the experimental signal/noise, and greatly simplify solvent-suppression by substantially reducing the water signal. We also present a tool, which allows filling solid-state NMR sample containers in a more efficient manner, greatly reducing the amount of supernatant water and maximizing signal/noise.

Proton assisted recoupling and protein structure determination.

We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between (13)C or (15)N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between (1)H-(13)C (or (1)H-(15)N) couplings to mediate zero quantum (13)C-(13)C (or (15)N-(15)N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C(1) (+/-)C(2) (-/+)H(Z) for (13)C-(13)C recoupling experiments (or N(1) (+/-)N(2) (-/+)H(Z) for (15)N-(15)N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with (13)C-(13)C spectra of uniformly (13)C, (15)N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2 x 10.4 kDa). The spectra, which were acquired at high MAS frequencies (omega(r)2pi>20 kHz) and magnetic fields (750-900 MHz (1)H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A) (13)C-(13)C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein.

3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints

In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 x 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 A.

13C spin dilution for simplified and complete solid-state NMR resonance assignment of insoluble biological assemblies.

A strategy for simplified and complete resonance assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-(13)C]- or [2-(13)C]glucose are very sparsely labeled, and the resulting 2D ssNMR spectra exhibit smaller line widths (by a factor of ∼2 relative to uniformly labeled proteins) and contain a reduced number of cross-peaks. This allows for an accelerated and straightforward resonance assignment without the necessity of time-consuming 3D spectroscopy or sophisticated pulse sequences. The strategy aims at complete backbone and side-chain resonance assignments based on bidirectional sequential walks. The approach was successfully demonstrated with the de novo assignment of the Type Three Secretion System PrgI needle protein. Using a limited set of simple 2D experiments, we report a 97% complete resonance assignment of the backbone and side-chain (13)C atoms.

The Molecular Organization of the Fungal Prion HET-s in Its Amyloid Form

The prion hypothesis states that it is solely the three-dimensional structure of the polypeptide chain that distinguishes the prion and nonprion forms of the protein. For HET-s, the atomic-resolution structure of the isolated prion domain HET-s(218-289), consisting of a highly ordered triangular cross-beta arrangement, is known. Here we present a solid-state NMR study of fibrils of the full-length HET-s prion in which we compare their spectra with spectra from isolated C-terminal prion domain fibrils and the crystalline N-terminal globular domain HET-s(1-227). The spectra reveal unequivocally that the highly ordered structure of the isolated prion domain HET-s(218-289) is conserved in the context of the full-length fibrils investigated here. However, the globular domain loses much of its tertiary structure while partly retaining its secondary structure, thus exhibiting behavior reminiscent of a molten globule. Flexible residues that may constitute the linker connecting the two domains are detected using INEPT (insensitive nuclei enhanced by polarization transfer) spectroscopy. Based on our data, we propose a structural model that is in line with a general model developed for amyloid fibrils built from a cross-beta core decorated with globular domains. The loss of structure in the HET-s globular domain sharply contrasts with the behavior observed for fibrils of Ure2p and suggests that there is considerable structural diversity in the fibrils of globular-domain-containing prions despite their similar appearances at the microscopic level.

Prion Fibrils of Ure2p Assembled under Physiological Conditions Contain Highly Ordered, Natively Folded Modules

The difference between the prion and the non-prion form of a protein is given solely by its three-dimensional structure, according to the prion hypothesis. It has been shown that solid-state NMR can unravel the atomic-resolution three-dimensional structure of prion fragments but, in the case of Ure2p, no highly resolved spectra are obtained from the isolated prion domain. Here, we demonstrate that the spectra of full-length fibrils of Ure2p interestingly lead to highly resolved solid-state NMR spectra. Prion fibrils formed under physiological conditions are therefore well-ordered objects on the molecular level. Comparing the full-length NMR spectra with the corresponding spectra of the prion and globular domains in isolation reveals that the globular part in particular shows almost perfect structural order. The NMR linewidths in these spectra are as narrow as the ones observed in crystals of the isolated globular domain. For the prion domain, the spectra reflect partial disorder, suggesting structural heterogeneity, both in isolation and in full-length Ure2p fibrils, although to different extents. The spectral quality is surprising in the light of existing structural models for Ure2p and in comparison to the corresponding spectra of the only other full-length prion fibrils (HET-s) investigated so far. This opens the exciting perspective of an atomic-resolution structure determination of the fibrillar form of a prion whose assembly is not accompanied by significant conformational changes and documents the structural diversity underlying prion propagation.

Structural investigations of molecular machines by solid-state NMR.

Essential biological processes such as cell motion, signaling,protein synthesis, and pathogen-host interactions rely on multifunctional molecular machines containing supramolecular assemblies, that is, noncovalently assembled protein subunits. Scientists would like to acquire a detailed atomic view of the complete molecular machine to understand its assembly process and functions. Structural biologists have used various approaches to obtain structural information such as X-ray crystallography, solution NMR, and electron microscopy. The inherent insolubility and large size of these multicomponent assemblies restrict the use of solution NMR, and their noncrystallinity and elongated shapes present obstacles to X-ray crystallography studies. Not limited by molecular weight or crystallinity, solid-state NMR (ssNMR) allows for structural investigations of supramolecular assemblies such as helical filaments, cross-β fibrils, or membrane-embedded oligomeric proteins. In this Account, we describe recent progress in the application of ssNMR to the elucidation of atomic structures of supramolecular assemblies. We highlight ssNMR methods to determine the subunit interfaces in symmetric arrangements. Our use of [1-(13)C]- or [2-(13)C]-glucose as a carbon source during bacterial protein expression results in significant (13)C spin dilution that drastically improves the spectral quality and enables us to detect meaningful structural restraints. Moreover, we can unequivocally determine intermolecular restraints using mixed [(1:1)1-(13)C/2-(13)C]-glucose labeled assemblies. We recently illustrated the power of this methodology with the structure determination of the type III secretion system (T3SS) needle. One crucial aspect in elucidating the atomic structure of these large multicomponent complexes is to determine the subunit-subunit interfaces. Notably, we could probe the needle subunit interfaces by collecting (13)C-(13)C intermolecular restraints. In contrast, these interfaces are not accessible via high-resolution cryo-EM. This approach is readily applicable to other supramolecular assemblies containing symmetrically repeating protein subunits, and could be combined with other techniques to get a more complete picture of multicomponent structures. To determine near-atomic structures of assemblies of biological interest, researchers could combine ssNMR data collected at the subunit interfaces with the envelope obtained from cryo-EM and potentially with monomeric subunit crystal structures.

Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoproteins interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection.