Backbone structure of Yersinia pestis Ail determined in micelles by NMR-restrained simulated annealing with implicit membrane solvation

Marassi, Francesca M.; Ding, Yi; Schwieters, Charles D.; Tian, Ye; Yao, Yong

doi:10.1007/s10858-015-9963-2

Cited by 26 publications

(31 citation statements)

References 48 publications

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“…A total of 100 structures were refined, starting from the coordinates of the 10 lowest energy structures calculated for Ail in micelles (PDB: 2N2L). 31 The 10 refined structures with lowest energy were selected for the final ensemble. The structural coordinates and experimental restraints have been deposited in the Protein Data Bank (PDB: 5VJ8).…”

Section: Methodsmentioning

confidence: 99%

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Structural Insights into the Yersinia pestis Outer Membrane Protein Ail in Lipid Bilayers

2017

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Yersinia pestis the causative agent of plague, is highly pathogenic and poses very high risk to public health. The outer membrane protein Ail (Adhesion invasion locus) is one of the most highly expressed proteins on the cell surface of Y. pestis, and a major target for the development of medical countermeasures. Ail is essential for microbial virulence and is critical for promoting the survival of Y. pestis in serum. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but the protein’s activity is influenced by the detergents in these samples, underscoring the importance of the surrounding environment for structure–activity studies. Here we describe the backbone structure of Ail, determined in lipid bilayer nanodiscs, using solution NMR spectroscopy. We also present solid-state NMR data obtained for Ail in membranes containing lipopolysaccharide (LPS), a major component of the bacterial outer membranes. The protein in lipid bilayers, adopts the same eight-stranded β-barrel fold observed in the crystalline and micellar states. The membrane composition, however, appears to have a marked effect on protein dynamics, with LPS enhancing conformational order and slowing down the 15N transverse relaxation rate. The results provide information about the way in which an outer membrane protein inserts and functions in the bacterial membrane.

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Section: Methodsmentioning

confidence: 99%

“…31 Ail folds as an eight-stranded β -barrel, with three short intracellular turns (T1–T3) and four long extracellular loops (EL1–EL4) that are significantly more flexible than the barrel body. The extracellular loops of Ail are important for function.…”

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Structural Insights into the Yersinia pestis Outer Membrane Protein Ail in Lipid Bilayers

2017

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“…The interactions of Ail with human host proteins are critical for promoting the survival of Y. pestis bacteria in serum, and are targets for therapy development. Two structures of Ail have been determined: by X-ray diffraction (Yamashita et al 2011) for the protein crystallized in tetra-ethylene glycol monooctyl ether (C8E4), and by solution NMR (Marassi et al 2015) for the protein in decyl-phosphocholine (DePC) micelles. Ail adopts the same eight-stranded β-barrel fold in both cases, but the protein’s activity is compromised by detergent (Ding et al 2015), which is present in both the crystal and micelle samples, underscoring the need to perform structure/function studies in detergent-free membranes.…”

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High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

et al. 2017

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The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40–0.60 ppm for 13C, 0.11–0.15 ppm for 1H, and 0.46–0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

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“… GB1 , protein G B1 domain (Gronenborn et al 1991; Kuszewski et al 1999); BAF , human barrier to autointegration factor, monomer (Cai et al 1998); ArfA - b, M. tuberculosis ammonia release facilitator A domain b (Teriete et al 2010); DHFR, L. casei apo dihydrofolate reductase (Feeney et al 2011); EIN , enzyme I N-terminal domain (Garrett et al 1997, 1999); fd coat , membrane bound form of fd bacteriophage pVIII coat protein (Marassi and Opella 2003); CrgA TM, M. tuberculosis cell division protein transmembrane region (Das et al 2015); OmpX, E. coli outer membrane protein X; input data include solution NMR restraints measured in nanodiscs (Hagn et al 2013) and solid-state NMR restraints measured in oriented bilayers (Mahalakshmi and Marassi 2008); Ail, Y. pestis attachment invasion locus outer membrane protein (Marassi et al 2015); ASR , Anabaena sensory rhodopsin, monomer (Wang et al 2013) a Defined as (i – i + 4) > 4 and including measurements of hydrogen exchange and paramagnetic relaxation enhancement b Dipolar coupling (DC) and chemical shift anisotropy (CSA) restraints, measured by solution NMR with proteins in weakly aligned media, or by solid-state NMR with protein in oriented bilayers …”

Section: Figmentioning

confidence: 99%

“…EEFx works with a dedicated force field that includes terms for van der Waals Lennard-Jones interatomic force, electrostatic force, and solvation free energy, and can be easily implemented with the wide range of experimental energy terms used for NMR structure determination. Notably, EEFx is very effective at guiding structure calculations towards the native state, even in the absence of large numbers of experimental measurements, and leads to significant improvements in structural quality, accuracy and precision for both soluble and membrane proteins (Cornilescu et al 2016; Jureka et al 2015; Lee et al 2016; Marassi et al 2015; Tian et al 2014, 2015). …”

Section: Introductionmentioning

confidence: 99%

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

et al. 2016

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Structure determination of proteins by NMR is unique in its ability to measure restraints, very accurately, in environments and under conditions that closely mimic those encountered in vivo. For example, advances in solid-state NMR methods enable structure determination of membrane proteins in detergent-free lipid bilayers, and of large soluble proteins prepared by sedimentation, while parallel advances in solution NMR methods and optimization of detergent-free lipid nanodiscs are rapidly pushing the envelope of the size limit for both soluble and membrane proteins. These experimental advantages, however, are partially squandered during structure calculation, because the commonly used force fields are purely repulsive and neglect solvation, Van der Waals forces and electrostatic energy. Here we describe a new force field, and updated energy functions, for protein structure calculations with EEFx implicit solvation, electrostatics, and Van der Waals Lennard-Jones forces, in the widely used program Xplor-NIH. The new force field is based primarily on CHARMM22, facilitating calculations with a wider range of biomolecules. The new EEFx energy function has been rewritten to enable OpenMP parallelism, and optimized to enhance computation efficiency. It implements solvation, electrostatics, and Van der Waals energy terms together, thus ensuring more consistent and efficient computation of the complete nonbonded energy lists. Updates in the related python module allow detailed analysis of the interaction energies and associated parameters. The new force field and energy function work with both soluble proteins and membrane proteins, including those with cofactors or engineered tags, and are very effective in situations where there are sparse experimental restraints. Results obtained for NMR-restrained calculations with a set of five soluble proteins and five membrane proteins show that structures calculated with EEFx have significant improvements in accuracy, precision, and conformation, and that structure refinement can be obtained by short relaxation with EEFx to obtain improvements in these key metrics. These developments broaden the range of biomolecular structures that can be calculated with high fidelity from NMR restraints.

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Backbone structure of Yersinia pestis Ail determined in micelles by NMR-restrained simulated annealing with implicit membrane solvation

Cited by 26 publications

References 48 publications

Structural Insights into the Yersinia pestis Outer Membrane Protein Ail in Lipid Bilayers

Structural Insights into the Yersinia pestis Outer Membrane Protein Ail in Lipid Bilayers

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

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