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[Display omitted] • Exact closed formulas for the influence of motion on nuclear Overhauser effects. • Isotropic, harmonic model of atom position fluctuations including steric repulsion. • Up to 20% underestimation of the true distance by motion for weak NOEs. • Small impact of motion for strong NOEs that correspond to small distances. • A B-factor derived from nuclear Overhauser effects. Nuclear Overhauser effects (NOEs) are influenced by motion. Here, we derive exact, analytical results for a model of isotropic, harmonic fluctuations of atom positions that corresponds to the one underlying crystallographic B-factors. The model includes steric repulsion and yields closed-form expressions for the expected value of general invertible functions of the distance between two atoms, with the special case r - 6 for NOEs. We discuss the implications for the definition of an NOE-based B-factor in solution NMR. [ABSTRACT FROM AUTHOR]

The nuclear magnetic resonance structure of the globular domain with residues 121–230 of a variant human prion protein with two disulfide bonds, hPrP(M166C/E221C), shows the same global fold as wild-type hPrP(121–230). It contains three α-helices of residues 144–154, 173–194 and 200–228, an anti-parallel β-sheet of residues 128–131 and 161–164, and the disulfides Cys166-Cys221 and Cys179-Cys214. The engineered extra disulfide bond in the presumed “protein X”-binding site is accommodated with slight, strictly localized conformational changes. High compatibility of hPrP with insertion of a second disulfide bridge in the protein X epitope was further substantiated by model calculations with additional variant structures. The ease with which the hPrP structure can accommodate a variety of locations for a second disulfide bond within the presumed protein X-binding epitope suggests a functional role for the extensive perturbation by a natural second disulfide bond of the corresponding region in the human doppel protein. [Copyright &y& Elsevier]

Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination. [Copyright &y& Elsevier]

Abstract: Homodimeric proteins pose a difficulty for NMR structure determination because the degeneracy of the chemical shifts in the two identical monomers implies an ambiguity in all assignments of distance restraints. For homodimeric proteins, residues involved in the interface between two monomers provide essential intermolecular NOEs. The structure determination of homodimeric proteins hence relies strongly on chemical shift assignments of these interface residues. Our paper discusses the influence of the extent of 1H chemical shift assignments of interface residues on the structure determinations of homodimeric proteins using the CYANA program. The results reveal that successful structure determinations of homodimeric proteins with automated NOE assignment depend on the percentage of assigned interface residues and that a high completeness of around 80–90% of the 1H chemical shift assignment in the interface is needed for reliable NMR structure determinations of homodimeric proteins for which no experimental distinction between intra- and intermolecular NOEs, e.g. by filtered NOESY experiments, is available. Our results also show that RMSD and target function values are insufficient to judge the quality of homodimeric structures determined using automated NOE assignment. Structure determinations of homodimeric proteins by NMR using conventional NOESY experiments are thus possible but more challenging than for monomeric proteins. [Copyright &y& Elsevier]

Abstract: Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as ZnII, CuI, and CdII that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the βE-domain of the early cysteine-labeled protein (Ec-1) from wheat (βE-Ec-1), which represents the first three-dimensional structure of a plant MT. The βE-domain comprises the 51 C-terminal residues of Ec-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear ZnII binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three ZnII ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys–metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by 15N relaxation experiments support the results of the structure determination in that βE-Ec-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced ZnII with CdII ions and examined the effects of metal ion release on incubation with a metal ion chelator. [Copyright &y& Elsevier]

The NMR structure of the recombinant human doppel protein, hDpl(24–152), contains a flexibly disordered “tail” comprising residues 24–51, and a globular domain extending from residues 52 to 149 for which a detailed structure was obtained. The globular domain contains four α-helices comprising residues 72–80 (α1), 101–115 (α2a), 117–121 (α2b), and 127–141 (α3), and a short two-stranded anti-parallel β-sheet comprising residues 58–60 (β1) and 88–90 (β2). The fold of the hDpl globular domain thus coincides nearly identically with the structure of the murine Dpl protein. There are close similarities with the human prion protein (hPrP) but, similar to the situation with the corresponding murine proteins, hDpl shows marked local differences when compared to hPrP: the β-sheet is flipped by 180° with respect to the molecular scaffold formed by the four helices, and the β1-strand is shifted by two residues toward the C terminus. A large solvent-accessible hydrophobic cleft is formed on the protein surface between β2 and α3, which has no counterpart in hPrP. The helix α2 of hPrP is replaced by two shorter helices, α2a and α2b. The helix α3 is shortened by more than two turns when compared with α3 of hPrP, which is enforced by the positioning of the second disulfide bond in hDpl. The C-terminal peptide segment 144–149 folds back onto the loop connecting β2 and α2. All but four of the 20 conserved residues in the globular domains of hPrP and hDpl appear to have a structural role in maintaining a PrP-type fold. The conservation of R76, E96, N110 and R134 in hDpl, corresponding to R148, E168, N183 and R208 in hPrP suggests that these amino acid residues might have essential roles in the so far unknown functions of PrP and Dpl in healthy organisms. [Copyright &y& Elsevier]

NMR spectra analysis for protein structure determination can now in many cases be performed by automated computational methods. This overview of the computational methods for NMR protein structure analysis presents recent automated methods for signal identification in multidimensional NMR spectra, sequence-specific resonance assignment, collection of conformational restraints, and structure calculation, as implemented in the CYANA software package. These algorithms are sufficiently reliable and integrated into one software package to enable the fully automated structure determination of proteins starting from NMR spectra without manual interventions or corrections at intermediate steps, with an accuracy of 1–2 Å backbone RMSD in comparison with manually solved reference structures. [ABSTRACT FROM AUTHOR]

The study of the spatial sampling of biomolecules is essential to understanding the structure–dynamics–function relationship. We have established a protocol for the determination of multiple-state ensembles based on exact measurements of the nuclear Overhauser effect (eNOE). The protocol is practical since it does not require any additional data, while all other NMR data sets must be supplemented by NOE restraints. The question arises as to how much structural and dynamics information is shared between the eNOEs and other NMR probes. We compile one of the largest and most diverse NMR data sets of a protein to date consisting of eNOEs, RDCs and J couplings for GB3. We show that the eNOEs improve the back-prediction of RDCs and J couplings, either upon use of more than one state, or in comparison to conventional NOEs. Our findings indicate that the eNOE data is self-consistent, consistent with other data, and that the structural representation with multiple states is warranted. [ABSTRACT FROM AUTHOR]

Despite their advantages in analysis, 4D NMR experiments are still infrequently used as a routine tool in protein NMR projects due to the long duration of the measurement and limited digital resolution. Recently, new acquisition techniques for speeding up multidimensional NMR experiments, such as nonlinear sampling, in combination with non-Fourier transform data processing methods have been proposed to be beneficial for 4D NMR experiments. Maximum entropy (MaxEnt) methods have been utilised for reconstructing nonlinearly sampled multi-dimensional NMR data. However, the artefacts arising from MaxEnt processing, particularly, in NOESY spectra have not yet been clearly assessed in comparison with other methods, such as quantitative maximum entropy, multidimensional decomposition, and compressed sensing. We compared MaxEnt with other methods in reconstructing 3D NOESY data acquired with variously reduced sparse sampling schedules and found that MaxEnt is robust, quick and competitive with other methods. Next, nonlinear sampling and MaxEnt processing were applied to 4D NOESY experiments, and the effect of the artefacts of MaxEnt was evaluated by calculating 3D structures from the NOE-derived distance restraints. Our results demonstrated that sufficiently converged and accurate structures (RMSD of 0.91 Å to the mean and 1.36 Å to the reference structures) were obtained even with NOESY spectra reconstructed from 1.6% randomly selected sampling points for indirect dimensions. This suggests that 3D MaxEnt processing in combination with nonlinear sampling schedules is still a useful and advantageous option for rapid acquisition of high-resolution 4D NOESY spectra of proteins. [ABSTRACT FROM AUTHOR]

The automated chemical shift assignment algorithm FLYA has been extended for use with stereo-array isotope labeled (SAIL) proteins to determine the sequence-specific resonance assignments of large proteins. Here we present the assignment of the backbone and sidechain chemical shifts of the 21 kDa thioldisulfide oxidoreductase DsbA from Escherichia coli that were determined with the SAIL-FLYA algorithm in conjunction with automated peak picking. No manual corrections of peak lists or assignments were applied. The assignments agreed with manually determined reference assignments in 95.4% of the cases if 16 input spectra were used, 94.1% if only 3D 13 C/ 15 N-resolved NOESY, CBCA(CO)NH, and 2D [ 13 C/ 15 N, 1 H]-HSQC were used, and 86.8% if exclusively 3D 13 C/ 15 N-resolved NOESY spectra were used. Considering only the assignments that are classified as reliable by the SAIL-FLYA algorithm, the degrees of agreement increased to 97.5%, 96.5%, and 94.2%, respectively. With our approach it is thus possible to automatically obtain almost complete and correct assignments of proteins larger than 20 kDa. [ABSTRACT FROM AUTHOR]

Highlights: [•] A perspective on the ensemble-based structure calculation using exact NOEs is given. [•] eNOEs can be used for ensemble-based structure calculations. [•] Structural ensembles may give insights into enzyme activity and protein folding. [•] Structural ensembles may give insights into dynamics including concerted motion. [Copyright &y& Elsevier]

Abstract: Selective autophagy requires the specific segregation of targeted proteins into autophagosomes. The selectivity is mediated by autophagy receptors, such as p62 and NBR1, which can bind to autophagic effector proteins (Atg8 in yeast, MAP1LC3 protein family in mammals) anchored in the membrane of autophagosomes. Recognition of autophagy receptors by autophagy effectors takes place through an LC3 interaction region (LIR). The canonical LIR motif consists of a WXXL sequence, N-terminally preceded by negatively charged residues. The LIR motif of NBR1 presents differences to this classical LIR motif with a tyrosine residue and an isoleucine residue substituting the tryptophan residue and the leucine residue, respectively. We have determined the structure of the GABARAPL-1/NBR1-LIR complex and studied the influence of the different residues belonging to the LIR motif for the interaction with several mammalian autophagy modifiers (LC3B and GABARAPL-1). Our results indicate that the presence of a tryptophan residue in the LIR motif increases the binding affinity. Substitution by other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation. This indicates that different LIRs can interact with autophagy modifiers with unique binding properties. [Copyright &y& Elsevier]

Abstract: The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69–162 of the 206-residue full-length protein forms a structure with a β1–β2–α1–β3–α2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an α-helix (α1), instead of a β-turn, is inserted between strand β2 and strand β3. A characteristic groove formed between α1 and the three-stranded antiparallel β-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function. [Copyright &y& Elsevier]

Abstract: The Notch signaling pathway is critical for many developmental processes and requires complex trafficking of both Notch receptor and its ligands, Delta and Serrate. In Drosophila melanogaster, the endocytosis of Delta in the signal-sending cell is essential for Notch receptor activation. The Neuralized protein from D. melanogaster (Neur) is a ubiquitin E3 ligase, which binds to Delta through its first neuralized homology repeat 1 (NHR1) domain and mediates the ubiquitination of Delta for endocytosis. Tom, a Bearded protein family member, inhibits the Neur-mediated endocytosis through interactions with the NHR1 domain. We have identified the domain boundaries of the novel NHR1 domain, using a screening system based on our cell-free protein synthesis method, and demonstrated that the identified Neur NHR1 domain had binding activity to the 20-residue peptide corresponding to motif 2 of Tom by isothermal titration calorimetry experiments. We also determined the solution structure of the Neur NHR1 domain by heteronuclear NMR methods, using a 15N/13C-labeled sample. The Neur NHR1 domain adopts a characteristic β-sandwich fold, consisting of a concave five-stranded antiparallel β-sheet and a convex seven-stranded antiparallel β-sheet. The long loop (L6) between the β6 and β7 strands covers the hydrophobic patch on the concave β-sheet surface, and the Neur NHR1 domain forms a compact globular fold. Intriguingly, in spite of the slight, but distinct, differences in the topology of the secondary structure elements, the structure of the Neur NHR1 domain is quite similar to those of the B30.2/SPRY domains, which are known to mediate specific protein–protein interactions. Further NMR titration experiments of the Neur NHR1 domain with the 20-residue Tom peptide revealed that the resonances originating from the bottom area of the β-sandwich (the L3, L5, and L11 loops, as well as the tip of the L6 loop) were affected. In addition, a structural comparison of the Neur NHR1 domain with the first NHR domain of the human KIAA1787 protein, which is from another NHR subfamily and does not bind to the 20-residue Tom peptide, suggested the critical amino acid residues for the interactions between the Neur NHR1 domain and the Tom peptide. The present structural study will shed light on the role of the Neur NHR1 domain in the Notch signaling pathway. [Copyright &y& Elsevier]

Abstract: Conjugation is a fundamental process for the rapid evolution of bacteria, enabling them, for example, to adapt to various environmental conditions or to acquire multi-drug resistance. NikA is one of the relaxosomal proteins that initiate the intercellular transfer of the R64 conjugative plasmid with the P-type origin of transfer, oriT. The three-dimensional structure of the N-terminal 51 residue fragment of NikA, NikA(1–51), which binds to the 17-bp repeat A sequence in R64 oriT, was determined by NMR to be a homodimer composed of two identical ribbon-helix-helix (RHH) domains, which are commonly found in transcriptional repressors. The structure determination of NikA(1–51) was achieved using automated NOE assignment with CYANA, without measuring filtered NOESY experiments to distinguish between the intra- and intermolecular NOEs, and without any a priori assumption on the tertiary or quaternary structure of the protein. Mutational experiments revealed that the DNA-binding region of the NikA(1–51) dimer is an anti-parallel β-sheet composed of one β-strand from each of the N-terminal ends of the two domains. Various biochemical experiments have indicated that the full length NikA(1–109) exists as a homotetramer formed through an α-helical domain at the C-terminus, and that the anti-parallel β-sheets of both dimeric domains bind to two homologous 5 bp internal repeats within repeat A. As a tetramer, the full length NikA(1–109) showed higher affinity to repeat A and bent the oriT duplex more strongly than NikA(1–51) did. Many RHH proteins are involved in specific DNA recognition and in protein–protein interactions. The discovery of the RHH fold in NikA suggests that NikA binds to oriT and interacts with the relaxase, NikB, which is unable to bind to the nick region in oriT without NikA. [Copyright &y& Elsevier]

Abstract: The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform 13C and 15N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution. [Copyright &y& Elsevier]

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(δ1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel β-barrel, with successive pairs of β-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded β-sheet beyond the height of the barrel. This protruding β-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies. [Copyright &y& Elsevier]

Calreticulin (CRT) is an abundant, soluble molecular chaperone of the endoplasmic reticulum. Similar to its membrane-bound homolog calnexin (CNX), it is a lectin that promotes the folding of proteins carrying N-linked glycans. Both proteins cooperate with an associated co-chaperone, the thiol-disulfide oxidoreductase ERp57. This enzyme catalyzes the formation of disulfide bonds in CNX and CRT-bound glycoprotein substrates. Previously, we solved the NMR structure of the central proline-rich P-domain of CRT comprising residues 189–288. This structure shows an extended hairpin topology, with three short anti-parallel β-sheets, three small hydrophobic clusters, and one helical turn at the tip of the hairpin. We further demonstrated that the residues 225–251 at the tip of the CRT P-domain are involved in direct contacts with ERp57. Here, we show that the CRT P-domain fragment CRT(221–256) constitutes an autonomous folding unit, and has a structure highly similar to that of the corresponding region in CRT(189–288). Of the 36 residues present in CRT(221–256), 32 form a well-structured core, making this fragment one of the smallest known natural sequences to form a stable non-helical fold in the absence of disulfide bonds or tightly bound metal ions. CRT(221–256) comprises all the residues of the intact P-domain that were shown to interact with ERp57. Isothermal titration microcalorimetry (ITC) now showed affinity of this fragment for ERp57 similar to that of the intact P-domain, demonstrating that CRT(221–256) may be used as a low molecular mass mimic of CRT for further investigations of the interaction with ERp57. We also solved the NMR structure of the 73-residue fragment CRT(189–261), in which the tip of the hairpin and the first β-sheet are well structured, but the residues 189–213 are disordered, presumably due to lack of stabilizing interactions across the hairpin. [Copyright &y& Elsevier]