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Chemical shift assignment is vital for nuclear magnetic resonance (NMR)-based studies of protein structures, dynamics, and interactions, providing crucial atomic-level insight. However, obtaining chemical shift assignments is labor intensive and requires extensive measurement time. To address this limitation, we previously proposed ARTINA, a deep learning method for automatic assignment of two-dimensional (2D)-4D NMR spectra. Here, we present an integrative approach that combines ARTINAwith AlphaFold and UCBShift, enabling chemical shift assignment with reduced experimental data, increased accuracy, and enhanced robustness for larger systems, as presented in a comprehensive study with more than 5000 automated assignment calculations on 89 proteins. We demonstrate that five 3D spectra yield more accurate assignments (92.59%) than pure ARTINA runs using all experimentally available NMR data (on average 10 3D spectra per protein, 91.37%), considerably reducing the required measurement time. We also showcase automated assignments of only 15Nlabeled samples, and report improved assignment accuracy in larger synthetic systems of up to 500 residues. [ABSTRACT FROM AUTHOR]

Summary Nuclear magnetic resonance (NMR) structures are represented by bundles of conformers calculated from different randomized initial structures using identical experimental input data. The spread among these conformers indicates the precision of the atomic coordinates. However, there is as yet no reliable measure of structural accuracy, i.e., how close NMR conformers are to the “true” structure. Instead, the precision of structure bundles is widely (mis)interpreted as a measure of structural quality. Attempts to increase precision often overestimate accuracy by tight bundles of high precision but much lower accuracy. To overcome this problem, we introduce a protocol for NMR structure determination with the software package CYANA, which produces, like the traditional method, bundles of conformers in agreement with a common set of conformational restraints but with a realistic precision that is, throughout a variety of proteins and NMR data sets, a much better estimate of structural accuracy than the precision of conventional structure bundles. [ABSTRACT FROM AUTHOR]

Background: The automation of objectively selecting amino acid residue ranges for structure superpositions is important for meaningful and consistent protein structure analyses. So far there is no widely-used standard for choosing these residue ranges for experimentally determined protein structures, where the manual selection of residue ranges or the use of suboptimal criteria remain commonplace. Results: We present an automated and objective method for finding amino acid residue ranges for the superposition and analysis of protein structures, in particular for structure bundles resulting from NMR structure calculations. The method is implemented in an algorithm, CYRANGE, that yields, without protein-specific parameter adjustment, appropriate residue ranges in most commonly occurring situations, including low-precision structure bundles, multi-domain proteins, symmetric multimers, and protein complexes. Residue ranges are chosen to comprise as many residues of a protein domain that increasing their number would lead to a steep rise in the RMSD value. Residue ranges are determined by first clustering residues into domains based on the distance variance matrix, and then refining for each domain the initial choice of residues by excluding residues one by one until the relative decrease of the RMSD value becomes insignificant. A penalty for the opening of gaps favours contiguous residue ranges in order to obtain a result that is as simple as possible, but not simpler. Results are given for a set of 37 proteins and compared with those of commonly used protein structure validation packages. We also provide residue ranges for 6351 NMR structures in the Protein Data Bank. Conclusions: The CYRANGE method is capable of automatically determining residue ranges for the superposition of protein structure bundles for a large variety of protein structures. The method correctly identifies ordered regions. Global structure superpositions based on the CYRANGE residue ranges allow a clear presentation of the structure, and unnecessary small gaps within the selected ranges are absent. In the majority of cases, the residue ranges from CYRANGE contain fewer gaps and cover considerably larger parts of the sequence than those from other methods without significantly increasing the RMSD values. CYRANGE thus provides an objective and automatic method for standardizing the choice of residue ranges for the superposition of protein structures. [ABSTRACT FROM AUTHOR]

A one-dimensional gas comprising N point particles undergoing elastic collisions within a finite space described by a Sinai billiard generating identical dynamical trajectories are calculated and analyzed with regard to strict extensivity of the entropy definitions of Boltzmann–Gibbs. Due to the collisions, trajectories of gas particles are strongly correlated and exhibit both chaotic and periodic properties. Probability distributions for the position of each particle in the one-dimensional gas can be obtained analytically, elucidating that the entropy in this special case is extensive at any given number N. Furthermore, the entropy obtained can be interpreted as a measure of the extent of interactions between molecules. The results obtained for the non-mixable one-dimensional system are generalized to mixable one- and two-dimensional systems, the latter by a simple example only providing similar findings. [ABSTRACT FROM AUTHOR]

Abstract: The WW domain is known as one of the smallest protein modules with a triple-stranded β-sheet fold. Here, we present the solution structure of the second WW domain from the mouse salvador homolog 1 protein. This WW domain forms a homodimer with a β-clam-like motif, as evidenced by size exclusion chromatography, analytical ultracentrifugation and NMR spectroscopy. While typical WW domains are believed to function as monomeric modules that recognize proline-rich sequences, by using conserved aromatic and hydrophobic residues that are solvent-exposed on the surface of the β-sheet, this WW domain buries these residues in the dimer interface. [Copyright &y& Elsevier]

NMR studies of amyloid β-peptides (Aβ) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer’s disease-related conformational polymorphism of Aβ. In the aqueous medium, neither of the polypeptides Aβ(1–40)ox or Aβ(1–42)ox (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of Aβ(1–40)ox and Aβ(1–42)ox are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in Aβ(1–42)ox when compared to Aβ(1–40)ox. The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that Aβ(1–42)ox in aqueous solution has much higher plaque-competence than Aβ(1–40)ox. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary We present NMRtist, an online platform that combines deep learning, large-scale optimization and cloud computing to automate protein NMR spectra analysis. Our website provides virtual storage for NMR spectra deposition together with a set of applications designed for automated peak picking, chemical shift assignment and protein structure determination. The system can be used by non-experts and allows protein assignments and structures to be determined within hours after the measurements, strictly without any human intervention. Availability and implementation NMRtist is freely available to non-commercial users at https://nmrtist.org. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (https://atb.uq.edu.au , last access: 17 February 2023) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of side chains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice, we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein–protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic biology and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally. [ABSTRACT FROM AUTHOR]

Most experimental methods for structural biology proceed in vitro and therefore the contribution of the intracellular environment on protein structure and dynamics is absent. Studying proteins at atomic resolution in living mammalian cells has been elusive due to the lack of methodologies. In-cell nuclear magnetic resonance spectroscopy (in-cell NMR) is an emerging technique with the power to do so. Here, we improved current methods of in-cell NMR by the development of a reporter system that allows monitoring the delivery of exogenous proteins into mammalian cells, a process that we called here "transexpression". The reporter system was used to develop an efficient protocol for in-cell NMR which enables spectral acquisition with higher quality for both disordered and folded proteins. With this method, the 3D atomic resolution structure of the model protein GB1 in human cells was determined with a backbone root-mean-square deviation (RMSD) of 1.1 Å. An improved method for in-cell NMR is developed using a reporter to evaluate protein delivery and delivery parameters to different cell lines and applied to IDPs and folded proteins including structure determination of GB1 at 1.1 Å resolution. [ABSTRACT FROM AUTHOR]

Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (atb.uq.edu.au) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of sidechains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein-protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally. [ABSTRACT FROM AUTHOR]

The Taiwan cobra (Naja naja atra) chymotrypsin inhibitor (NACI) consists of 57 amino acids and is related to other Kunitz-type inhibitors such as bovine pancreatic trypsin inhibitor (BPTI) and Bungarus fasciatus fraction IX (BF9), another chymotrypsin inhibitor. Here we present the solution structure of NACI. We determined the NMR structure of NACI with a root-mean-square deviation of 0.37 Å for the backbone atoms and 0.73 Å for the heavy atoms on the basis of 1,075 upper distance limits derived from NOE peaks measured in its NOESY spectra. To investigate the structural characteristics of NACI, we compared the three-dimensional structure of NACI with BPTI and BF9. The structure of the NACI protein comprises one 310-helix, one α-helix and one double-stranded antiparallel β-sheet, which is comparable with the secondary structures in BPTI and BF9. The RMSD value between the mean structures is 1.09 Å between NACI and BPTI and 1.27 Å between NACI and BF9. In addition to similar secondary and tertiary structure, NACI might possess similar types of protein conformational fluctuations as reported in BPTI, such as Cys14-Cys38 disulfide bond isomerization, based on line broadening of resonances from residues which are mainly confined to a region around the Cys14-Cys38 disulfide bond. [ABSTRACT FROM AUTHOR]

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, protondetected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atomspecific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution. [ABSTRACT FROM AUTHOR]

Proteins are inherently dynamic systems whose motions cover large ranges in both magnitude and timescale. Because of the omnipresence of motion, it is likely that dynamics have important roles in the function of biomolecules. For detailed understanding of a protein's function, the three-dimensional structure and description of its dynamics are therefore required. Structure determination methods are well established, and NMR-relaxation phenomena provide insights into local molecular dynamics; moreover, recently several attempts have been made to detect concerted motion. Here, we present an ensemble-based structure-determination protocol using ensemble-averaged distance restraints obtained from exact NOE rates. Application to the model protein GB3 establishes an ensemble of structures that reveals correlated motion across the ?-sheet, concerted motion between the backbone and side chains localized in the structure core, and a lack of concerted conformational exchange between the ?-sheet and the ?-helix. [ABSTRACT FROM AUTHOR]

In the bacterium Azotobacter vinelandii, a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7) has been identified. These epimerases are responsible for the epimerization of β-D-mannuronic acid to α-L-guluronic acid in alginate polymers. The epimerases consist of two types of structural modules, designated A (one or two copies) and R (one to seven copies). The structure of the catalytically active A-module from the smallest epimerase AlgE4 (consisting of AR) has been solved recently. This paper describes the NMR structure of the R-module from AlgE4 and its titration with a substrate analogue and paramagnetic thulium ions. The R-module folds into a right-handed parallel β-roll. The overall shape of the R-module is an elongated molecule with a positively charged patch that interacts with the substrate. Titration of the R-module with thulium indicated possible calcium binding sites in the loops formed by the nonarepeat sequences in the N-terminal part of the molecule and the importance of calcium binding for the stability of the R-module. Structure calculations showed that calcium ions can be incorporated in these loops without structural violations and changes. Based on the structure and the electrostatic surface potential of both the A- and R-module from AlgE4, a model for the appearance of the whole protein is proposed. [ABSTRACT FROM AUTHOR]

The concept of metal chaperones involves transient binding of metallic cofactors by specific proteins for delivery to enzymes in which they function. Metal chaperones thus provide a protective, as well as a transport, function. We report the first structure of a heme chaperone, CcmE, which comprises these two functions. We propose that the covalent attachment of heme to an exposed histidine occurs after heme binding at the surface of a rigid molecule with a flexible C-terminal domain. CcmE belongs to a family of proteins with a specific fold, which all share a function in delivery of specific molecular cargo. [Copyright &y& Elsevier]

Escherichia coli diacylglycerol kinase (DGK) is an integral membrane protein, which catalyses the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It is a unique trimeric enzyme, which does not share sequence homology with typical kinases. It exhibits a notable complexity in structure and function despite of its small size. Here, chemical shift assignment of wild-type DGK within lipid bilayers was carried out based on 3D MAS NMR, utilizing manual and automatic analysis protocols. Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, we could detect that the nucleotide substrate induces a substantial conformational change, most likely directing DGK into its catalytic active form. Furthermore, functionally relevant interprotomer interactions are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays. [ABSTRACT FROM AUTHOR]

A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale. [ABSTRACT FROM AUTHOR]

The p63 gene encodes amaster regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the C-terminal domain of the p63 gene can cause ankyloblepharonectodermal defects-cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility and severe, long-lasting skin erosions. Despite deep knowledge of p63 functions, little is known about mechanisms underlying disease pathology and possible treatments. Here, we show that multiple AEC-associated p63 mutations, but not those causative of other diseases, lead to thermodynamic protein destabilization, misfolding, and aggregation, similar to the known p53 gain-of-function mutants found in cancer. AEC mutant proteins exhibit impaired DNA binding and transcriptional activity, leading to dominant negative effects due to coaggregation with wild-type p63 and p73. Importantly, p63 aggregation occurs also in a conditional knock-in mouse model for the disorder, in which the misfolded p63 mutant protein leads to severe epidermal defects. Variants of p63 that abolish aggregation of the mutant proteins are able to rescue p63's transcriptional function in reporter assays as well as in a human fibroblast-to-keratinocyte conversion assay. Our studies reveal that AEC syndrome is a protein aggregation disorder and opens avenues for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and theWW domain of Pin1, and finally discuss our current research and future directions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aβ(1–40) and Aβ(1–42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aβ(1–42) is the more neurotoxic species, aggregatesmuch faster, and dominates in senile plaque of Alzheimer’s disease (AD) patients. Although small Aβ oligomers are believed to be the neurotoxic species, Aβ amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a diseaserelevant Aβ(1–42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15–42 forming a double-horseshoe–like cross–β-sheet entity with maximally buried hydrophobic side chains. Residues 1–14 are partially ordered and in a β-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure. [ABSTRACT FROM AUTHOR]

Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination. [ABSTRACT FROM AUTHOR]

The inclusion of peptoid monomers into antimicrobial peptides (AMPs) increases their proteolytic resistance, but introduces conformational flexibility (reduced hydrogen bonding ability and cis/trans isomerism). We here use NMR spectroscopy to answer how the insertion of a peptoid monomer influences the structure of a regular α‐helical AMP upon interaction with a dodecyl phosphocholine (DPC) micelle. Insertion of [(2‐methylpropyl)amino]acetic acid in maculatin‐G15 shows that the structural change and conformational flexibility depends on the site of insertion. This is governed by the micelle interaction of the amphipathic helices flanking the peptoid monomer and the side chain properties of the peptoid and its preceding residue.A high‐resolution structure of a peptoid–peptide hybrid (“peptomer”) is presented. The peptoid residue breaks the helix and displays cis/trans isomerism. Maculatin‐Nleu11 displays only the trans isomer. Maculatin‐Nleu13 displays almost equimolar amounts of cis and trans conformers. Peptide amphipathicity and peptoid hydrophobicity govern the cis/trans isomerism. [ABSTRACT FROM AUTHOR]

Allostery and correlated motion are key elements linking protein dynamics with the mechanisms of action of proteins. Here, we present PDBCor, an automated and unbiased method for the detection and analysis of correlated motions from experimental multi-state protein structures. It uses torsion angle and distance statistics and does not require any structure superposition. Clustering of protein conformers allows us to extract correlations in the form of mutual information based on information theory. With PDBcor, we elucidated correlated motion in the WW domain of PIN1, the protein GB3, and the enzyme cyclophilin, in line with reported findings. Correlations extracted with PDBcor can be utilized in subsequent assays including nuclear magnetic resonance (NMR) multi-state structure optimization and validation. As a guide for the interpretation of PDBcor results, we provide a series of protein structure ensembles that exhibit different levels of correlation, including non-correlated, locally correlated, and globally correlated ensembles. [Display omitted] • PDBcor algorithm extracts protein-correlated motion from protein ensembles • PDBcor is based on GMM clustering and information theory • High sensitivity to correlated motion comes from the use of protein distances • PDBcor is unbiased, as the structure superposition step is not required Ashkinadze et al. present an unbiased algorithm, PDBcor, for the extraction of protein-correlated motion from protein structural ensembles. Using clustering and mutual information, this algorithm is based on the statistical analysis of protein interresidual distances. The authors validate it on three model proteins with known structural correlations [ABSTRACT FROM AUTHOR]

Repeat proteins are built of modules, each of which constitutes a structural motif. We have investigated whether fragments of a designed consensus armadillo repeat protein (ArmRP) recognize each other. We examined a split ArmRP consisting of an N-capping repeat (denoted Y), three internal repeats (M), and a C-capping repeat (A). We demonstrate that the C-terminal MA fragment adopts a fold similar to the corresponding part of the entire protein. In contrast, the N-terminal YM2 fragment constitutes a molten globule. The two fragments form a 1:1 YM2:MA complex with a nanomolar dissociation constant essentially identical to the crystal structure of the continuous YM3A protein. Molecular dynamics simulations show that the complex is structurally stable over a 1 μs timescale and reveal the importance of hydrophobic contacts across the interface. We propose that the existence of a stable complex recapitulates possible intermediates in the early evolution of these repeat proteins. [ABSTRACT FROM AUTHOR]

Summary: Phosphopantetheine transferases represent a class of enzymes found throughout all forms of life. From a structural point of view, they are subdivided into three groups, with transferases from group II being the most widespread. They are required for the posttranslational modification of carrier proteins involved in diverse metabolic pathways. We determined the crystal structure of the group II phosphopantetheine transferase Sfp from Bacillus in complex with a substrate carrier protein in the presence of coenzyme A and magnesium, and observed two protein-protein interaction sites. Mutational analysis showed that only the hydrophobic contacts between the carrier protein’s second helix and the C-terminal domain of Sfp are essential for their productive interaction. Comparison with a similar structure of a complex of human proteins suggests that the mode of interaction is highly conserved in all domains of life. [Copyright &y& Elsevier]

Background Simple peak-picking algorithms, such as those based on lineshape fitting, perform well when peaks are completely resolved in multidimensional NMR spectra, but often produce wrong intensities and frequencies for overlapping peak clusters. For example, NOESY-type spectra have considerable overlaps leading to significant peak-picking intensity errors, which can result in erroneous structural restraints. Precise frequencies are critical for unambiguous resonance assignments. Results To alleviate this problem, a more sophisticated peaks decomposition algorithm, based on non-negative matrix factorization (NMF), was developed. We produce peak shapes from Fourier-transformed NMR spectra. Apart from its main goal of deriving components from spectra and producing peak lists automatically, the NMF approach can also be applied if the positions of some peaks are known a priori, e.g. from consistently referenced spectral dimensions of other experiments. Conclusions Application of the NMF algorithm to a three-dimensional peak list of the 23kDa bi-domain section of the RcsD protein (RcsD-ABL-HPt, residues 688-890) as well as to synthetic HSQC data shows that peaks can be picked accurately also in spectral regions with strong overlap. [ABSTRACT FROM AUTHOR]

Summary: As methods for analysis of biomolecular structure and dynamics using nuclear magnetic resonance spectroscopy (NMR) continue to advance, the resulting 3D structures, chemical shifts, and other NMR data are broadly impacting biology, chemistry, and medicine. Structure model assessment is a critical area of NMR methods development, and is an essential component of the process of making these structures accessible and useful to the wider scientific community. For these reasons, the Worldwide Protein Data Bank (wwPDB) has convened an NMR Validation Task Force (NMR-VTF) to work with wwPDB partners in developing metrics and policies for biomolecular NMR data harvesting, structure representation, and structure quality assessment. This paper summarizes the recommendations of the NMR-VTF, and lays the groundwork for future work in developing standards and metrics for biomolecular NMR structure quality assessment. [ABSTRACT FROM AUTHOR]

Adequate digital resolution and signal sensitivity are two critical factors for protein structure determinations by solution NMR spectroscopy. The prime objective for obtaining high digital resolution is to resolve peak overlap, especially in NOESY spectra with thousands of signals where the signal analysis needs to be performed on a large scale. Achieving maximum digital resolution is usually limited by the practically available measurement time. We developed a method utilizing non-uniform sampling for balancing digital resolution and signal sensitivity, and performed a large-scale analysis of the effect of the digital resolution on the accuracy of the resulting protein structures. Structure calculations were performed as a function of digital resolution for about 400 proteins with molecular sizes ranging between 5 and 33 kDa. The structural accuracy was assessed by atomic coordinate RMSD values from the reference structures of the proteins. In addition, we monitored also the number of assigned NOESY cross peaks, the average signal sensitivity, and the chemical shift spectral overlap. We show that high resolution is equally important for proteins of every molecular size. The chemical shift spectral overlap depends strongly on the corresponding spectral digital resolution. Thus, knowing the extent of overlap can be a predictor of the resulting structural accuracy. Our results show that for every molecular size a minimal digital resolution, corresponding to the natural linewidth, needs to be achieved for obtaining the highest accuracy possible for the given protein size using state-of-the-art automated NOESY assignment and structure calculation methods. [ABSTRACT FROM AUTHOR]

Abstract: The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains. [Copyright &y& Elsevier]

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.Wealso 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. [ABSTRACT FROM AUTHOR]

Summary: Nuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data. [ABSTRACT FROM AUTHOR]

Summary: The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent “blind” assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR. [ABSTRACT FROM AUTHOR]

Summary: The Rcs-signaling system is one of the most remarkable phosphorelay pathways in Enterobacteriaceae, comprising several membrane-bound and soluble proteins. Within the complex phosphotransfer pathway, the histidine phosphotransferase (HPt) domain of the RcsD membrane-bound component serves as a crucial factor in modulating the phosphorylation state of the transcription factor RcsB. We have identified a new domain, RcsD-ABL, located N terminally to RcsD-HPt that interacts with RcsB as well. We have determined its structure, characterized its interaction interface with RcsB, and built a structural model of the complex of the RcsD-ABL domain with RcsB. Our results indicate that the effector domain of RcsB, which normally binds to DNA, is recognized by RcsD-ABL, whereas the HPt domain interacts with the phosphoreceiver domain of RcsB. [ABSTRACT FROM AUTHOR]

n in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC = 7.7 μM and 37.9 μM, respectively) and the related West Nile virus protease (IC = 6.3 μM and 39.0 μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters. [ABSTRACT FROM AUTHOR]

Summary: The zinc finger CW (zf-CW) domain is a motif of about 60 residues that is frequently found in proteins involved in epigenetic regulation. Here, we determined the NMR solution structure of the zf-CW domain of the human zf-CW and PWWP domain containing protein 1 (ZCWPW1). The zf-CW domain adopts a new fold in which a zinc ion is coordinated tetrahedrally by four conserved Cys ligand residues. The tertiary structure of the zf-CW domain partially resembles that adopted by the plant homeo domain (PHD) finger bound to the histone tail, suggesting that the zf-CW domain and the PHD finger have similar functions. The solution structure of the complex of the zf-CW domain with the histone H3 tail peptide (1-10) with trimethylated K4 clarified its binding mode. Our structural and biochemical studies have identified the zf-CW domain as a member of the histone modification reader modules for epigenetic regulation. [ABSTRACT FROM AUTHOR]

The γ-secretase complex has a decisive role in the development of Alzheimer's disease, in that it cleaves a precursor to create the amyloid β peptide whose aggregates form the senile plaques encountered in the brains of patients. Γ-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many, of the mutations encountered in early onset familial Alzheimer's disease are linked to presenilin 1, the catalytic component of γ-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1. but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop. [ABSTRACT FROM AUTHOR]

Investigating proteins ‘at work’ in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments. [ABSTRACT FROM AUTHOR]

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform 13C/15N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using thecyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three β-sheets. [ABSTRACT FROM AUTHOR]

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds. Type I thioesterases (TEI) catalyse the release step from the assembly line of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain. [ABSTRACT FROM AUTHOR]

Recent advances in cell-free expression protocols have opened a new avenue toward high-resolution structural investigations of membrane proteins by x-ray crystallography and NMR spectroscopy. One of the biggest challenges for liquid-state NMR-based structural investigations of membrane proteins is the significant peak overlap in the spectra caused by large line widths and limited chemical shift dispersion of a-helical proteins. Contributing to the limited chemical shift dispersion is the fact that ≈6O% of the amino acids in transmembrane regions consist of only six different amino acid types. This principle disadvantage, however, can be exploited to aid in the assignment of the backbone resonances of membrane proteins; by [sup15]N/[sup13]C-double-labeling of these six amino acid types, sequential connectivities can be obtained for large stretches of the transmembrane segments where number and length of stretches consisting exclusively of these six amino acid types are enhanced compared with the remainder of the protein. We show by experiment as well as by statistical analysis that this labeling scheme provides a large number of sequential connectivities in transmembrane regions and thus constitutes a tool for the efficient assignment of membrane protein backbone resonances. [ABSTRACT FROM AUTHOR]

Summary: The SF3a complex, consisting of SF3a60, SF3a66, and SF3a120, in 17S U2 snRNP is crucial to spliceosomal assembly. SF3a120 contains two tandem SURP domains (SURP1 and SURP2), and SURP2 is responsible for binding to SF3a60. We found that the SURP2 fragment forms a stable complex with an SF3a60 fragment (residues 71–107) and solved its structure by NMR spectroscopy. SURP2 exhibits a fold of the α1-α2-310-α3 topology, and the SF3a60 fragment forms an amphipathic α helix intimately contacting α1 of SURP2. We also solved the SURP1 structure, which has the same fold as SURP2. The protein-binding interface of SURP2 is quite similar to the corresponding surface of SURP1, except for two amino acid residues. One of them, Leu169, is characteristic of SF3a120 SURP2 among SURP domains. Mutagenesis showed that this single Leu residue is the critical determinant for complex formation, which reveals the protein recognition mechanism in the subunit assembly. [Copyright &y& Elsevier]

Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination. [ABSTRACT FROM AUTHOR]

The NMR structures of the recombinant prion proteins from chicken (Gallus gallus; chPrP), the red-eared slider turtle (Trachemys scripta; tPrP), and the African clawed frog (Xenopus !aevis; xlPrP) are presented. The amino acid sequences of these prion proteins show ∼30% identity with mammalian prion proteins. All three species form the same molecular architecture as mammalian PrPc, with a long, flexibly disordered tail attached to the N-terminal end of a globular domain. The globular domain in chPrP and tPrP contains three α-helices, one short 310-helix, and a short antiparallel β-sheet. In xlPrP, the globular domain includes three a-helices and a somewhat longer fl-sheet than in the other species. The spatial arrangement of these regular secondary structures coincides closely with that of the globular domain in mammalian prion proteins. Based on the low sequence identity to mammalian PrPs, comparison of chPrP, tPrP, and xlPrP with mammalian PrV structures is used to identify a set of essential amino acid positions for the preservation of the same PrPc fold in birds, reptiles, amphibians, and mammals. There are additional conserved residues without apparent structural roles, which are of interest for the ongoing search for physiological functions of PrPc in healthy organisms. [ABSTRACT FROM AUTHOR]

The NMR structures of the recombinant cellular form of the prion proteins (PrP9 of the cat (Felis catus), dog (canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented In all of these species. PrV consists of an N-terminal flexibly extended tail with ∼100 amino acid residues and a C-terminal globular domain of ∼100 residues with three a-helices and a short antiparallel β-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrV structures, there are local differences between the globular domains of the different species. Because the five newly determined PrPC structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrPC three- dimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies. [ABSTRACT FROM AUTHOR]

The multisubunit elongation factor 1 (eEF1) is required for the elongation step of eukaryotic protein synthesis. The eEF1 complex consists of four subunits: eEF1A, a G-protein that shuttles aminoacylated tRNAs to the ribosome; eEF1Bα and eEF1Bβ, two guanine nucleotide exchange factors, and eEF1Bγ. Although its exact function remains unknown, this latter subunit is present in all eukaryotes. Recombinant human eEF1Bγ has been purified and shown to consist of two independent domains. We have utilized high resolution NMR to determine the three-dimensional structure of the 19 kDa C-terminal fragment (domain 2). The structure consists of a five-stranded anti-parallel β-sheet surrounded by α-helices and resembles a contact lens. Highly conserved residues are mainly located on the concave face, suggesting thereby that this side of the molecule might be involved in some biologically relevant interface(s). Although the isolated domain 2 appears to be mostly monomeric in solution, biochemical and structural data indicate a potential homodimer. The proposed dimer model can be further positioned within the quaternary arrangement of the whole eEF1 assembly. [ABSTRACT FROM AUTHOR]

The nuclear magnetic resonance structure of the unliganded pheromone-binding protein (PBP) from Bombyx mori at pH above 6.5, BmPBPB, consists of seven helices with residues 3–8, 16–22, 29–32, 46–59, 70–79, 84–100, and 107–124, and contains the three disulfide bridges 19–54, 50–108, and 97–117. This polypeptide fold encloses a large hydrophobic cavity, with a sufficient volume to accommodate the natural ligand bombykol. The polypeptide folds in free BmPBPB and in crystals of a BmPBP–bombykol complex are nearly identical, indicating that the B-form of BmPBP in solution represents the active conformation for ligand binding. [Copyright &y& Elsevier]