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We compiled an NMR data set consisting of exact nuclear Overhauser enhancement (eNOE) distance limits, residual dipolar couplings (RDCs) and scalar (J) couplings for GB3, which forms one of the largest and most diverse data set for structural characterization of a protein to date. All data have small experimental errors, which are carefully estimated. We use the data in the research article Vogeli et al., 2015, Complementarity and congruence between exact NOEs and traditional NMR probes for spatial decoding of protein dynamics, J. Struct. Biol., 191, 3, 306–317, doi:10.1016/j.jsb.2015.07.008 [1] for cross-validation in multiple-state structural ensemble calculation. We advocate this set to be an ideal test case for molecular dynamics simulations and structure calculations.
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.
A B-factor for NOEs?
(2022)
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.
NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.
An automated NMR chemical shift assignment algorithm was developed using multi-objective optimization techniques. The problem is modeled as a combinatorial optimization problem and its objective parameters are defined separately in different score functions. Some of the heuristic approaches of evolutionary optimization are employed in this problem model. Both, a conventional genetic algorithm and multi-objective methods, i.e., the non-dominated sorting genetic algorithms II and III (NSGA2 and NSGA3), are applied to the problem. The multi-objective approaches consider each objective parameter separately, whereas the genetic algorithm followed a conventional way, where all objectives are combined in one score function. Several improvement steps and repetitions on these algorithms are performed and their combinations are also created as a hyper-heuristic approach to the problem. Additionally, a hill-climbing algorithm is also applied after the evolutionary algorithm steps. The algorithms are tested on several different datasets with a set of 11 commonly used spectra. The test results showed that our algorithm could assign both sidechain and backbone atoms fully automatically without any manual interactions. Our approaches could provide around a 65% success rate and could assign some of the atoms that could not be assigned by other methods.
Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cβ, C′) resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.
To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on ‘indirect’ structural information from peak patterns and chemical shift changes but not ‘direct’ data explicitly including interatomic distances and angles. To fully understand the functions and physical properties of proteins inside cells, it is indispensable to obtain explicit structural data or determine three-dimensional (3D) structures of proteins in cells. Whilst the short lifetime of cells in a sample tube, low sample concentrations, and massive background signals make it difficult to observe NMR signals from proteins inside cells, several methodological advances help to overcome the problems. Paramagnetic effects have an outstanding potential for in-cell structural analysis. The combination of a limited amount of experimental in-cell data with software for ab initio protein structure prediction opens an avenue to visualise 3D protein structures inside cells. Conventional nuclear Overhauser effect spectroscopy (NOESY)-based structure determination is advantageous to elucidate the conformations of side-chain atoms of proteins as well as global structures. In this article, we review current progress for the structure analysis of proteins in living systems and discuss the feasibility of its future works.
In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity “Cambrian-like” [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger “Ordovician-Silurian” fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.