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ARIA 2.0 provides a new user interface for streamlined project management. The complete set of program and protocol settings can now be modified graphically.
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The assignment of nuclear Overhauser effect (NOE) resonances is the crucial step in determining the three-dimensional structure of biomolecules from nuclear magnetic resonance (NMR) data. Our program, Ambiguous Restraints for Iterative Assignment (ARIA), treats Noe assignment as an integral part of the structure determination process. This chapter...
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... rationalize and simplify the setup of an ARIA run, version 2.0 offers a new GUI that replaces the HTML-based interface used in previous versions (see Fig. 1). All relevant program and protocol parameters can be adjusted via the GUI. Default values are given if appropriate and a pop-up help menu explains the meaning of each ...
Citations
... 53,57 Both lists were further simplified by discarding restraints that had more than 20 possible assignments, as this was shown, in the context of solution NMR, to have the potential to improve the quality of the calculated model. 58,59 An attempt to discard restraints that had more than five possible assignments resulted in failure to obtain a converged structure. An initial list of ADRs and non-ambiguous restraints was obtained by aggregating both restraint lists (fully and sparsely labeled) while giving precedence to restraints arising from the inherently less ambiguous, more informative sparse data, in spectral regions where the two peak-lists overlapped (see details regarding generation of distance restraints in the SI). ...
F-specific filamentous phages, elongated particles with circular single-stranded DNA encased in a symmetric protein capsid, undergo an intermediate step, where thousands of homodimers of a non-structural protein, gVp, bind to newly synthesized strands of DNA, preventing further DNA replication and preparing the circular genome in an elongated conformation for assembly of a new virion structure at the membrane. While the structure of the free homodimer is known, the ssDNA-bound conformation has yet to be determined. We report an atomic-resolution structure of the gVp monomer bound to ssDNA of fd phage in the nucleoprotein complex elucidated via magic-angle spinning solid-state NMR. The model presents significant conformational changes with respect to the free form. These modifications facilitate the binding mechanism and possibly promote cooperative binding in the assembly of the gVp–ssDNA complex.
... Structural restraints and structure calculation. Based on the almost complete assignment of the 1 H, 15 59 and automatically assigned and converted into NOE distance restraints using ARIA 2.3 75,76 in an iterative procedure. All resulting NOE assignments were inspected manually. ...
(Macro-)autophagy is a compartmental degradation pathway conserved from yeast to mammals. The yeast protein Atg8 mediates membrane tethering/hemifusion and cargo recruitment and is essential for autophagy. The human MAP1LC3/GABARAP family proteins show high sequence identity with Atg8, but MAP1LC3C is distinguished by a conspicuous amino-terminal extension with unknown functional significance. We have determined the high-resolution three-dimensional structure and measured the backbone dynamics of MAP1LC3C by NMR spectroscopy. From Ser18 to Ala120, MAP1LC3C forms an α-helix followed by the ubiquitin-like tertiary fold with two hydrophobic binding pockets used by MAP1LC3/GABARAP proteins to recognize targets presenting LC3-interacting regions (LIRs). Unlike other MAP1LC3/GABARAP proteins, the amino-terminal region of MAP1LC3C does not form a stable helix α1 but a “sticky arm” consisting of a polyproline II motif on a flexible linker. Ser18 at the interface between this linker and the structural core can be phosphorylated in vitro by protein kinase A, which causes additional conformational heterogeneity as monitored by NMR spectroscopy and molecular dynamics simulations, including changes in the LIR-binding interface. Based on these results we propose that the amino-terminal polyproline II motif mediates specific interactions with the microtubule cytoskeleton and that Ser18 phosphorylation modulates the interplay of MAP1LC3C with its various target proteins.
... Peak intensities derived from the NOE experiments mentioned above and backbone dihedral angle restraints derived from TALOS-N [44] were introduced into a ARIA2.3 structure calculation protocol [60,61] using standard options. The RDC values (HN and NC ′) for regions of defined secondary structure were introduced using the SANI option starting from the fifth ARIA iteration. ...
Eukaryotic elongation factor 2 kinase (eEF-2K), an atypical calmodulin-activated protein kinase, regulates translational elongation by phosphorylating its substrate, eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome. The activation and activity of eEF-2K are critical for survival under energy-deprived conditions and is implicated in a variety of essential physiological processes. Previous biochemical experiments have indicated that the binding site for the substrate eEF-2 is located in the C-terminal domain of eEF-2K, a region predicted to harbor several α-helical repeats. Here, using NMR methodology, we have determined the solution structure of a C-terminal fragment of eEF-2K, eEF-2K562–725 that encodes two α-helical repeats. The structure of eEF-2K562–725 shows signatures characteristic of TPR domains and of their SEL1-like sub-family. Furthermore, using the analyses of NMR spectral perturbations and ITC measurements, we have localized the eEF-2 binding site on eEF-2K562–725. We find that eEF-2K562–725 engages eEF-2 with an affinity comparable to that of the full-length enzyme. Furthermore, eEF-2K562–725 is able to inhibit the phosphorylation of eEF-2 by full-length eEF-2K in trans. Our present studies establish that eEF-2K562–725 encodes the major elements necessary to enable the eEF-2K/eEF-2 interactions.
... Interresidue N i -H N •••• O = C j ′ hydrogen bonds were detected by the observation of h3 J NC′ couplings in a 2D long-range HNCO experiment 54 . Version 2.3.2 of ARIA (Ambiguous Restraints for Iterative Assignment) was used for NOESY cross peak assignment and structure calculation 35,36 . NOE cross peak assignments of the acquired NOESY spectra were obtained by an iterative procedure using a combination of manual and automatic steps. ...
Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5′, of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop–end-helix interface.
... The NOE assignment was completed using ARIA2.3 (46), concomitantly with structure calculation. The ARIA program was run with eight iterations including twenty structures and a final iteration including 100 structures. ...
p65 is a member of the NF-κB family of transcriptional regulatory proteins that functions as the activating component of the p65-p50 heterodimer. Through its acidic transactivation domain (TAD), p65 has the capacity to form interactions with several different transcriptional regulatory proteins, including TFIIB, TFIIH, CREB-binding protein (CBP)/p300 and TAFII31. Like other acidic TADs, the p65 TAD contains two subdomains (p65TA1 and p65TA2) that interact with different regulatory factors depending on the target gene. Despite its role in controlling numerous NF-κB target genes, there are no high-resolution structures of p65TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65TA1 transitions into a helical conformation that includes its characteristic ΦXXΦΦ motif (Φ = hydrophobic amino acid). Structural and functional studies demonstrate that the two binding interfaces are primarily stabilized by three hydrophobic amino acids within the ΦXXΦΦ motif and these residues are also crucial to its ability to activate transcription. Taken together, the results provide an atomic level description of how p65TA1 is able to bind different transcriptional regulatory factors needed to activate NF-κB target genes.
... Structure calculations were performed using ARIA2.3 (CNS1.2) following published protocols 41 . Briefly, TALOS+ 42 was used to obtain dihedral angle constraints from chemical shifts ( 1 H, 1 Hα, 13 Cα, 13 Cβ, 13 C and 15 N), and peak intensities of the 3D NOESY experiments were introduced directly into ARIA to derive ambiguous distance restraints. ...
Eukaryotic elongation factor 2 kinase (eEF-2K) phosphorylates its only known physiological substrate, elongation factor 2 (eEF-2), which reduces the affinity of eEF-2 for the ribosome and results in an overall reduction in protein translation rates. The C-terminal region of eEF-2K, that is predicted to contain several SEL-1 like helical repeats (SLRs), is required for the phosphorylation of eEF-2. Using solution NMR methodology, we have determined the structure of a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K627-725) that encompasses a region previously suggested to be essential for eEF-2 phosphorylation. eEF-2K627-725 contains four helices, of which the first (I) is flexible, and does not pack stably against the ordered helical core formed by the last three helices (II-IV). The helical core shows significant structural similarity with members of the tetratricopeptide repeat (TPR) family that includes SLRs. The two penultimate helices, II and III, comprise the TPR, and the final helix, IV, appears to have a capping function. The eEF-2K627-725 structure illustrates that the C-terminal deletion that was shown to abolish eEF-2 phosphorylation does so by destabilizing IV and therefore, the helical core. Indeed, mutation of two conserved C-terminal tyrosines (Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation, leads to the unfolding of eEF-2K627-725. Preliminary functional analyses indicate that neither a peptide encoding a region deemed crucial for eEF-2 binding, nor isolated eEF-2K627-725 inhibit eEF-2 phosphorylation by full-length eEF-2K. Taken together, our data suggest that the extreme C-terminal region of eEF-2K, in isolation, does not provide a primary docking site for eEF-2.
... The structure of the Ca 2+ -CaM•eEF-2K CBD complex was performed using ARIA2.3 (CNS2.1.2) (Habeck et al., 2004) using the PARALLHDG force field with PROLSQ nonbonded energy terms (Engh and Huber, 1991) and experimental distance and angular restraints. Details of the structure calculation and analysis are described in the Supplemental Experimental Procedures. ...
Binding of Ca2+-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca2+ from the CaM C-lobe sites, even under high Ca2+ conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells.
... Structures were calculated using ambiguously assigned distance restraints derived from crosspeaks picked in 3D 13 C-and 15 N-edited nuclear Overhauser effect spectroscopy-HSQC spectra (Marion et al., 1989;Vranken et al., 2005) recorded at 298 K. Phi and psi dihedral angle restraints for residues in regular secondary structure elements predicted by DANGLE (Cheung et al., 2010) were applied in the high-temperature dynamics phases of each structure calculation and omitted during the final cooling stage. In the final iteration of the ARIA 2.3-driven calculations (Habeck et al., 2004), a total of 5,626 restraints were used to generate 100 structures from which an ensemble of the 20 structures which agree most closely with the experimental restraints was selected and refined in a full force field including explicit solvent (Linge et al., 2003). Structure quality parameters are summarized in Table 1. ...
Escherichia coli possesses a number of specific K(+) influx and efflux systems that maintain an appropriate intracellular K(+) concentration. Although regulatory mechanisms have been identified for a number of these transport systems, the exact mechanism through which K(+) concentration is sensed in the cell remains unknown. In this work we show that Kbp (K(+) binding protein, formerly YgaU), a soluble 16-kDa cytoplasmic protein from Escherichia coli, is a highly specific K(+) binding protein and is required for normal growth in the presence of high levels of external K(+). Kbp binds a single potassium ion with high specificity over Na(+) and other metal ions found in biological systems, although, in common with K(+) transporters, it also binds Rb(+) and Cs(+). Dissection of the K(+) binding determinants of Kbp suggests a mechanism through which Kbp is able to sense changes in K(+) concentration over the relevant range of intracellular K(+) concentrations.
... Distance restraints were derived from NOESY (nuclear Overhauser effect spectroscopy) cross-peaks with the initial mapping from normalized intensity to distance and grouped in distance bins. NOE distance restraints were incorporated in restrained MD calculations using the ambiguous distance restraints formalism [47] using ARIA 2.3 [48] and CNS [49]. Loose backbone dihedral restraints for regions of regular secondary structure predicted based on secondary chemical shifts by DANGLE [50] were incorporated during the high temperature phases of the simulations but omitted during the final cooling phase. ...
... The N-terminal helices, 3 10 (residues 3-5), α2 (8-11) and α3 (16)(17)(18)(19)(20)(21)(22)(23)(24) are co-planar with the C-terminal helices α9 (126-135), α10 (137-144) and α11 (147-152) and together with one of the longest helices, α6 (57-75), form one large face of the wedge. The other two long helices, α7 (79-99) and α8 (107-122), are co-planar with α5 (45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55) and form the other large face. These two faces enclose an internal cavity that is closed at the thick end of the wedge by α4 (29)(30)(31)(32)(33)(34)(35)(36)(37), which is almost perpendicular to the long axis of the molecule. ...
Fatty acid and retinol binding proteins (FARs) comprise a family of unusual α-helix rich lipid binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein (Ce-FAR-7) is from a subfamily of FARs that does not appear to be important at the host-parasite interface. We have therefore examined Na-FAR-1 from the blood-feeding intestinal parasite of humans, Necator americanus . The three dimensional structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, respectively, reveals an a-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand binding cavity and an additional C-terminal a-helix. Titration of apo -Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein:ligand complexes can be formed. Na-FAR-1, and possibly other FARs, may have a wider repertoire for hydrophobic ligand binding, as confirmed here by our finding that a range of neutral and polar lipids co-purify with the bacterial recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.
Copyright 2015 The Author(s).
... We also note that YAPP differs from other automated assignment protocols like CYANA or ARIA [12,13] in one important aspect. The YAPP protocol is not aimed at finding a unique NOE assignment, but treats the assignments as variables. ...
We present an analysis of the results obtained at CASD-NMR round 3 by the CHESHIRE and the YAPP methods. To determine protein structures, the CHESHIRE method uses solely information provided by NMR chemical shifts, while the YAPP method uses an automated assignment of NOESY spectra. Of the ten targets of CASD-NMR round 3, nine CHESHIRE predictions and eight YAPP ones were submitted. The eight YAPP predictions ranged from 0.7 to 1.9 Å Cα accuracy, with an average of 1.3 Å. The nine CHESHIRE predictions ranged from 0.8 to 2.6 Å Cα accuracy for the ordered regions of the proteins, with an average of 1.6 Å. Taken together, these results illustrate how the NOESY based YAPP method and the chemical shift based CHESHIRE method can provide structures of comparable quality.
