Structure d’une nanomachine bactérienne

Loquet, Antoine; Habenstein, Birgit; Demers, Jean‐Philippe; Becker, Stefan; Lange, Adam

doi:10.1051/medsci/20122811008

Cited by 4 publications

(5 citation statements)

References 10 publications

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“…The T3SS, or injectisome, is a supramolecular assembly found in Gram-negative bacteria, such as Shigella , Salmonella , Escherichia , Pseudomonas or Yersinia 10 , which serves to deliver toxic effector proteins into their target eukaryotic host cell during infection 11 , 12 . The current study extends and improves on our previous work focusing on T3SS needles, which include the atomic model of Salmonella typhimurium 13 – 15 using ssNMR data and helical parameters from scanning transmission electron microscopy (STEM), the ssNMR resonance assignment of Shigella flexneri MxiH needles 16 which allowed to identify a common architecture for T3SS needles, the study of dynamics and conformational heterogeneity of MxiH needles using DNP-enhanced ssNMR 17 , and solid-state NMR assignment strategies using sparsely 13 C-labeled samples 18 – 20 or highly deuterated proteins 21 . The new hybrid approach combines a 7.7 Å cryo-EM density map reconstructed using 100,000 needle segment images 22 with extensive structural information obtained from ssNMR: 162 backbone dihedral angles and 996 carbon-carbon distance constraints from proton-driven spin diffusion (PDSD 23 , 24 ) experiments.…”

Section: Introductionsupporting

confidence: 72%

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

et al. 2014

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We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state NMR (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the Type-III Secretion System (T3SS) needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent dataset of 691 ssNMR constraints and STEM measurements. The hybrid model resolves the conformation of the non-conserved N-terminus, that occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.

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Section: Introductionsupporting

confidence: 72%

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

et al. 2014

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“…8−17 We recently reported a complete 3D atomic model of a biological assembly in its filamentous state, the type III secretion system (T3SS) needle. 18,19 The T3SS needle is formed by the helical repetition of a single protein subunit (named PrgI for the Salmonella typhimurium T3SS). Structural modeling of this system was complicated due to the presence of three different unique intermolecular interfaces (Figure 1A,B): an axial (between subunits i and i+11) and two lateral interfaces (between i and i +5 or i and i+6, respectively).…”

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confidence: 99%

“…Solid-state NMR spectroscopy (ssNMR) provides a powerful complementary method to X-ray crystallography, solution NMR, and EM for the investigation of biological molecular machines in their assembled state . Atomic information that can be extracted from ssNMR data opens the way to characterize the structure, interactions, and dynamics of biomolecular complexes of growing complexity. − We recently reported a complete 3D atomic model of a biological assembly in its filamentous state, the type III secretion system (T3SS) needle. , The T3SS needle is formed by the helical repetition of a single protein subunit (named PrgI for the Salmonella typhimurium T3SS). Structural modeling of this system was complicated due to the presence of three different unique intermolecular interfaces (Figure A,B): an axial (between subunits i and i+11) and two lateral interfaces (between i and i+5 or i and i+6, respectively).…”

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confidence: 99%

Atomic Structure and Handedness of the Building Block of a Biological Assembly

et al. 2013

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Noncovalent supramolecular assemblies possess in general several unique subunit−subunit interfaces.The basic building block of such an assembly consists of several subunits and contains all unique interfaces. Atomic-resolution structures of monomeric subunits are typically accessed by crystallography or solution NMR and fitted into electron microscopy density maps. However, the structure of the intact building block in the assembled state remains unknown with this hybrid approach. Here, we present the solid-state NMR atomic structure of the building block of the type III secretion system needle. The building block structure consists of a homotetrameric subunit complex with three unique supramolecular interfaces. Side-chain positions at the interfaces were solved at atomic detail. The high-resolution structure reveals unambiguously the helical handedness of the assembly, determined to be right-handed for the type III secretion system needle.Additionally, the axial rise per subunit could be extracted from the tetramer structure and independently validated by mass-per-length measurements. M any biological macromolecular assemblies, such as filaments, fibrils, or capsids, consist of multiple copies of protein subunits in a symmetrical arrangement. The building block of these assemblies is defined as the smallest set of subunits that contains all unique subunit−subunit interfaces, similar to the concept of the asymmetric unit in crystallography. The structure determination of supramolecular assemblies remains a challenging task, mostly due to the noncrystallinity that severely restricts the use of X-ray crystallography. The fitting of crystal subunit structures into density maps obtained from electron-microscopy (EM) offers nowadays 1−4 a suitable approach to obtain three-dimensional (3D) models of biological assemblies. Nevertheless, despite major improvements in EM methodology, density maps with a resolution sufficient to decipher atomic details about individual subunits in the assembly are still very scarce. 5,6 Solid-state NMR spectroscopy (ssNMR) provides a powerful complementary method to X-ray crystallography, solution NMR, and EM for the investigation of biological molecular machines in their assembled state.7 Atomic information that can be extracted from ssNMR data opens the way to characterize the structure, interactions, and dynamics of biomolecular complexes of growing complexity. 8−17 We recently reported a complete 3D atomic model of a biological assembly in its filamentous state, the type III secretion system (T3SS) needle.18,19 The T3SS needle is formed by the helical repetition of a single protein subunit (named PrgI for the Salmonella typhimurium T3SS). Structural modeling of this system was complicated due to the presence of three different unique intermolecular interfaces ( Figure 1A,B): an axial (between subunits i and i+11) and two lateral interfaces (between i and i +5 or i and i+6, respectively). The smallest building block that needs to be considered in order to reconstruct an atom...

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“…As neither solubility nor crystallinity is required for solidstate nuclear magnetic resonance (ssNMR) studies, large filamentous structures, such as bacteriophages, 5,6 the HIV capsid, 7,8 or the secretion system needles 2,9 are directly accessible to magic-angle spinning (MAS) ssNMR structural investigations at atomic detail. Over the past decade, several studies of supramolecular assemblies using ssNMR have been reported.…”

Section: Introductionmentioning

confidence: 99%

Structural Investigations of Molecular Machines by Solid-State NMR

2013

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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.

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Structure d’une nanomachine bactérienne

Cited by 4 publications

References 10 publications

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

Atomic Structure and Handedness of the Building Block of a Biological Assembly

Structural Investigations of Molecular Machines by Solid-State NMR

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