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Structure determination of complex molecular machines requires a combination of an increasing number of experimental methods with highly specialized software geared toward each data source to properly handle the gathered data. Recently, we introduced the two software packages PowerFit and DisVis. These combine high-resolution structures of atomic subunits with density maps from cryo-electron microscopy or distance restraints, typically acquired by chemical cross-linking coupled with mass spectrometry, respectively. Here, we report on recent advances in both GPGPU-accelerated software packages: PowerFit is a tool for rigid body fitting of atomic structures in cryo-electron density maps and has been updated to also output reliability indicators for the success of fitting, through the use of the Fisher z-transformation and associated confidence intervals; DisVis aims at quantifying the information content of distance restraints and identifying false-positive restraints. We extended its analysis capabilities to include an analysis of putative interface residues and to output an average shape representing the putative location of the ligand. To facilitate their use by a broad community, they have been implemented as web portals harvesting both local CPU resources and GPGPU-accelerated EGI grid resources. They offer user-friendly interfaces, while minimizing computational requirements, and provide a first interactive view of the results. The portals can be accessed freely after registration via http://milou.science.uu.nl/services/DISVIS and http://milou.science.uu.nl/services/POWERFIT., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the serine protease inhibitor of Kazal-type (SPINK) family. SPINK6 was shown to be expressed in human stratum corneum and is able to inhibit several KLKs such as KLK4, -5, -12, -13 and -14. In order to understand the structural traits of the specific inhibition we solved the structure of SPINK6 in solution by NMR-spectroscopy and studied its interaction with KLKs. Thereby, beside the conserved binding mode, we identified an alternate binding mode which has so far not been observed for SPINK inhibitors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We present an updated and integrated version of our widely used protein-protein docking and binding affinity benchmarks. The benchmarks consist of non-redundant, high-quality structures of protein-protein complexes along with the unbound structures of their components. Fifty-five new complexes were added to the docking benchmark, 35 of which have experimentally measured binding affinities. These updated docking and affinity benchmarks now contain 230 and 179 entries, respectively. In particular, the number of antibody-antigen complexes has increased significantly, by 67% and 74% in the docking and affinity benchmarks, respectively. We tested previously developed docking and affinity prediction algorithms on the new cases. Considering only the top 10 docking predictions per benchmark case, a prediction accuracy of 38% is achieved on all 55 cases and up to 50% for the 32 rigid-body cases only. Predicted affinity scores are found to correlate with experimental binding energies up to r=0.52 overall and r=0.72 for the rigid complexes., (Copyright © 2015. Published by Elsevier Ltd.)

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions., (Copyright © 2014. Published by Elsevier Ltd.)

Availability of high-resolution atomic structures is one of the prerequisites for a mechanistic understanding of biomolecular function. This atomic information can, however, be difficult to acquire for interesting systems such as high molecular weight and multi-subunit complexes. For these, low-resolution and/or sparse data from a variety of sources including NMR are often available to define the interaction between the subunits. To make best use of all the available information and shed light on these challenging systems, integrative computational tools are required that can judiciously combine and accurately translate the sparse experimental data into structural information. In this Perspective we discuss NMR techniques and data sources available for the modeling of large and multi-subunit complexes. Recent developments are illustrated by particularly challenging application examples taken from the literature. Within this context, we also position our data-driven docking approach, HADDOCK, which can integrate a variety of information sources to drive the modeling of biomolecular complexes. It is the synergy between experimentation and computational modeling that will provides us with detailed views on the machinery of life and lead to a mechanistic understanding of biomolecular function., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac-O1 and Lac-nonoperator complexes. Structural analysis of the Lac-O1 and Lac-O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac-O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein-DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac-nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac-O3 complex is the same as in the Lac-O1 and Lac-O2 complexes, as judged from the results of hydrogen/deuterium experiments.

The low-density lipoprotein receptor-related protein (LRP) interacts with more than 30 ligands of different sizes and structures that can all be replaced by the receptor-associated protein (RAP). The double module of complement type repeats, CR56, of LRP binds many ligands including all three domains of RAP and alpha2-macroglobulin, which promotes the catabolism of the Abeta-peptide implicated in Alzheimer's disease. To understand the receptor-ligand cross-talk, the NMR structure of CR56 has been solved and ligand binding experiments with RAP domain 1 (RAPd1) have been performed. From chemical shift perturbations of both binding partners upon complex formation, a HADDOCK model of the complex between CR56 and RAPd1 has been obtained. The binding residues are similar to a common binding motif suggested from alpha2-macroglobulin binding studies and provide evidence for an understanding of their mutual cross-competition pattern. The present structural results convey a simultaneous description of both binding partners of an LRP-ligand complex and open a route to a broader understanding of the binding specificity of the LRP receptor, which may involve a general four-residue receptor-ligand recognition motif common to all LRP ligands. The present result may be beneficial in the design of antagonists of ligand binding to the LDL receptor family, and especially of drugs for treatment of Alzheimer's disease.

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.

The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

Recent technological advances in NMR spectroscopy have alleviated the size limitations for the determination of biomolecular structures in solution. At the same time, novel NMR parameters such as residual dipolar couplings are providing greater accuracy. As this review shows, the structures of protein-protein and protein-nucleic acid complexes up to 50 kDa can now be accurately determined. Although de novo structure determination still requires considerable effort, information on interaction surfaces from chemical shift perturbations is much easier to obtain. Advances in modelling and data-driven docking procedures allow this information to be used for determining approximate structures of biomolecular complexes. As a result, a wealth of information has become available on the way in which proteins interact with other biomolecules. Of particular interest is the fact that these NMR-based methods can be applied to weak and transient protein-protein complexes that are difficult to study by other structural methods.

The ubiquitination pathway is the main pathway for protein degradation in eukaryotic cells. The attachment of ubiquitin to a substrate protein is catalyzed by three types of enzymes, namely a ubiquitin activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Here, the structure of the human ubiquitin-conjugating enzyme (E2) UbcH5B has been solved by a combination of homology modeling, NMR relaxation data and automated NOE assignments. Comparison to E2 structures solved previously by X-ray crystallography or NMR shows in all cases the same compact fold, but differences are observed in the orientation of both N and C-terminal alpha-helices. The N-terminal helix that is involved in binding to ubiquitin ligases (E3) displays a different position, which could have consequences for precise E2-E3 recognition. In addition, multiple conformations of the side-chain of Asn77 are found in solution, which contrasts the single hydrogen-bonded conformation in the crystal structures of E2 enzymes. The possible implication of this conformational freedom of Asn77 for its catalytic function is discussed.

Specific protein-protein interactions play crucial roles in the regulation of any biological process. Recently, a new protein-protein interaction domain termed PB1 (Phox and Bem1) was identified, which is conserved throughout evolution and present in diverse proteins functioning in signal transduction, cell polarity and survival. Here, we investigated the structure and molecular interactions of the PB1 heterodimer complex composed of the PB1 domains of the yeast proteins Bem1 and Cdc24. A structural model of the Cdc24 PB1 was built by homology modeling and molecular dynamics simulations, and experimentally validated by 15N nuclear Overhauser effect spectroscopy (NOESY)-heteronuclear single quantum coherence (HSQC) analysis. Residues at the interface of the complex for both proteins were identified by NMR titration experiments. A model of the heterodimer was obtained by docking of the two PB1 domains with HADDOCK, which applies ambiguous interaction restraints on residues at the interface to drive the docking procedure. The refined model was validated by site-directed mutagenesis on both Bem1 and Cdc24. Finally, the docking was repeated from the newly published NMR structure of Cdc24, allowing us to assess the performance of homology-based docking. Our results provide insight into the molecular structure of the Bem1-Cdc24 PB1-mediated heterodimer, which allowed identification of critical residues at the binding interface.

The high sensitivity of modern NMR instrumentation, in combination with full deuteration, enabled the measurement of long-range NOEs between amide protons in a fully deuterated protein corresponding to distances up to 8A. These are beyond the limit normally observed in protonated samples. Such long-distance NOEs could be observed using long mixing times, which became possible due to reduced spin diffusion and T1 relaxation of the amide protons in the fully deuterated sample. This information was used in combination with secondary structure restraints derived from secondary chemical shifts for structure calculations. With these backbone amide proton NOEs only, a unique fold could be obtained with positional root mean square deviations from the average of 1.30 and 2.25 A for backbone and heavy atoms, respectively. Despite the low density of restraints, no mirror image problems were observed. Addition of sidechain NOE information increased the precision of the ensemble and in particular of the core packing. The structures obtained in this way were close to the published crystal structure. NOE completeness analysis revealed that the cumulative completeness is still more than 80% for an 8.0 A cut-off distance.

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.

The glucocorticoid receptor (GR) is a hormone dependent nuclear receptor that regulates gene transcription when bound to the glucocorticoid response element (GRE). The GRE acts as an allosteric effector, inducing a structural change in the GR DNA-binding domain (GR DBD) upon binding, thereby switching the GR to an active conformation. A similar conformational change can be induced by two single point mutations: Ser459Ala and Pro493Arg. Structural and dynamical aspects of the conformational switch have been investigated by molecular dynamics simulations. Our results indicate that these two mutants, which share a similar phenotype, exert their action at a structural level through different mechanisms. In the Arg(493) mutant, the D-loop and the second helix are stabilized in a conformation that preforms the protein-protein dimer interface. In the Ala(459) mutant, the structurally important hydrogen bond between Arg(496) and Ser(459) is missing, which leads to a core reorganization and a reorientation of the second helical region. Although remote, both in sequence and three dimensional structure, these two mutations induce structural changes that are ultimately reflected in similar regions of the GR DBD structure, namely the D-loop and the short second helical region. These correspond to hot area of the GR DBD that are important both for DNA-binding and for the proper formation of the protein-protein interface. The conformational rearrangements in these area are proposed to decrease unfavorable protein-DNA and protein-protein contacts and allow unspecific DNA-binding leading to the squelching phenotype of the mutants. The GR DBD can thus exist in two states, a transcriptionally active and an inactive state. Switching between these states can be accomplished either by GRE binding or by the described mutations., (Copyright 2003 Elsevier Science Ltd.)

The hydration of the collagen-like Ac-(Gly-Pro-Hyp)(6)-NH(2) triple-helical peptide in solution was investigated using an integrated set of high-resolution NMR hydration experiments, including different recently developed exchange-network editing methods. This approach was designed to explore the hydration dynamics in the proximity of labile groups, such as the hydroxyproline hydroxyl group, and revealed that the first shell of hydration in collagen-like triple helices is kinetically labile with upper limits for water molecule residence times in the nanosecond to sub-nanosecond range. This result is consistent with a "hopping" hydration model in which solvent molecules are exchanged in and out of solvation sites at a rate that is not directly correlated to the degree of site localization. The hopping model thus reconciles the dynamic view of hydration revealed by NMR with the previously suggested partially ordered semi-clathrate-like cylinder of hydration. In addition, the nanosecond to sub-nanosecond upper limits for water molecule residence times imply that hydration-dehydration events are not likely to be the rate-limiting step for triple helix self-recognition, complementing previous investigations on water dynamics in collagen fibers. This study has also revealed labile proton features expected to facilitate the characterization of the structure and folding of triple helices in collagen peptides., (Copyright 2000 Academic Press.)

The stability and (un)folding of the 19-residue peptide, SCVTLYQSWRYSQADNGCA, corresponding to the first beta-hairpin (residues 10 to 28) of the alpha-amylase inhibitor tendamistat (PDB entry 3AIT) has been studied by molecular dynamics simulations in explicit water under periodic boundary conditions at several temperatures (300 K, 360 K and 400 K), starting from various conformations for simulation lengths, ranging from 10 to 30 ns. Comparison of trajectories of the reduced and oxidized native peptides reveals the importance of the disulphide bridge closing the beta-hairpin in maintaining a proper turn conformation, thereby insuring a proper side-chain arrangement of the conserved turn residues. This allows rationalization of the conservation of those cysteine residues among the family of alpha-amylase inhibitors. High temperature simulations starting from widely different initial configurations (native beta-hairpin, alpha and left-handed helical and extended conformations) begin sampling similar regions of the conformational space within tens of nanoseconds, and both native and non-native beta-hairpin conformations are recovered. Transitions between conformational clusters are accompanied by an increase in energy fluctuations, which is consistent with the increase in heat capacity measured experimentally upon protein folding. The folding events observed in the various simulations support a model for beta-hairpin formation in which the turn is formed first, followed by hydrogen bond formation closing the hairpin, and subsequent stabilization by side-chain hydrophobic interactions., (Copyright 2000 Academic Press.)

The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2 corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(+/-19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(+/-11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(+/-4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3' and O4' and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA., (Copyright 1998 Academic Press.)

The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2H and 17O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 degreesC. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/operator complexes shows very similar structural environments for the water molecules mediating specific contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition., (Copyright 1998 Academic Press.)

The solution structure of the DNA binding domain of lac repressor (headpiece 1-56; HP56) has been refined using data from 2D and 3D NMR spectroscopy. The structure was derived from 1546 restraints (giving an average of 27.6 per residue), comprising 389 intraresidual, 402 sequential, 385 medium range and 325 long range distance restraints and also 30 phi and 15 chi 1 dihedral angle restraints. The structures were determined by the method of direct refinement against nuclear Overhauser enhancement peak volumes with the program DINOSAUR. The final set of 32 selected structures displayed an r.m.s. deviation from the average of 0.43(+/-0.08) A angstroms (backbone) and 0.95(+/-0.08) angstroms (all heavy atoms) for the best defined region of the protein (residues 3 to 49). The ensemble R-factor was 0.35, which indicates close correspondence with the experimental data. The structures revealed good stereochemical qualities. The conformations of the NMR structures of free and DNA complexed lac repressor headpiece were compared. The regions comprising the secondary structure elements show close correspondence for both conformations. However, the conformation of the loop between helix II and III changes considerably upon complexation of the headpiece. This change in the conformation of the loop in lac HP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lac operator DNA. Finally, the lac headpiece residues that are intolerant to mutations were analysed. Most of these mutation-sensitive residues are important for a correct folding of the headpiece region, and a number of these residues are also involved in contacting the operator DNA.

In structure determination by X-ray crystallography and solution NMR spectroscopy, experimental data are collected as time and ensemble-averages. Thus, in principle, appropriate time and ensemble-averaged models should be used. Refinement of an ensemble of conformers rather than one single structure against the experimental NMR data could, however, result in overfitting the data because of the significantly increased number of parameters. To avoid overfitting, complete cross-validation, which provides an unbiased measure of the fit, has been applied to nuclear Overhauser effect derived distance refinement. Using two synthetic test cases, a correlation was demonstrated between the cross-validated measure to the fit (defined in terms of root-mean-square deviations from the distance restraints and number of violations) and the number of models that best reproduce the conformational variability in solution. A new method, based on a probability map, has been used to generate good representations of the resulting ensembles of structures. The method has also been applied to observed NMR data for two proteins, interleukin 4 and interleukin 8. For interleukin 4, cross-validation indicates that a single-conformer model gives the most accurate representation of the structure, whereas conventional measures of fit between the experimental data and those calculated from the model decrease when increasing the number of conformers, indicating overfitting. For interleukin 8, complete cross-validation predicts a twin-conformer model to be the most faithful representation of the experimental data. Two distinct conformations for the loop formed by residues 16 to 22 emerge from the family of twin-conformer structures. The putative alternate conformation of the loop is not observed in the crystal structure of interleukin 8. However, because of crystal packing contacts in this region this does not necessarily exclude the presence of the alternate conformation in solution. The twin-conformer model is supported by observed chemical exchange line broadening for the amide of His18 obtained by 15N relaxation studies. This region has also been implied to be involved in receptor binding.

The solution structure of the glucocorticoid receptor (GR) DNA-binding domain (DBD), consisting of 93 residues, has been refined from two and three-dimensional NMR data using an ensemble iterative relaxation matrix approach followed by direct NOE refinement with DINOSAUR. A set of 47 structures of the rat GR fragment Cys440-Arg510 was generated with distance geometry and further refined with a combination of restrained energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an extensive set of NOE build-up curves in H2O and 2H2O via relaxation matrix calculations (1186 distance constraints from NOE intensities, 10 phi and 22 chi 1 dihedral angle constraints from J- coupling data were used for the calculations). The root-mean-square deviation values of the 11 best structures on the well-determined part of the protein (Cys440 to Ser448, His451 to Glu469 and Pro493 to Glu508) are 0.60 A and 1.20 A from the average for backbone and all heavy atoms, respectively. The final structures have R-factors around 0.40 and good stereochemical qualities. The first zinc-coordinating domain of the GR DBD is very similar to the crystal structure with a root-mean-square difference of 1.4 A. The second zinc-coordinating domain is still disordered in solution. No secondary structure element is found in this domain in the free state. As suggested by crystallographic studies on the estrogen receptor DBD-DNA and GR DBD-DNA complexes, part of this region will form a distorted helix and the D-box will undergo a conformational change upon cooperative binding to DNA.

The Arc repressor of Salmonella bacteriophage P22 is a dimeric sequence-specific DNA-binding protein. The solution structure of Arc has been determined from 2D NMR data using an "ensemble" iterative relaxation matrix approach (IRMA) followed by direct NOE refinement with DINOSAUR. A set of 51 structures was generated with distance geometry and further refined with a combination of restrained energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an extensive set of NOE build-ups in H2O and 2H2O via relaxation matrix calculations from the ensemble of structures. Methyl group rotation, aromatic ring flaps and internal mobility effects (via order parameters obtained from a free molecular dynamics run in water) were included in these calculations. The best structures were finally refined with direct NOE constraints following a slow-cooling simulated annealing protocol. In this final refinement stage, theoretical NOE intensities were directly compared with the experimental data and forces were derived using a simple two-spin approximation for the gradient of the NOE function. Dynamic assignment was applied to the peaks involving unassigned diastereotopic groups. The structure is determined to a precision (r.m.s.d. from the average excluding the ill defined C and N-terminal region) of 0.55 and 1.10 A for backbone and all atoms, respectively. The final structures, with R factor values around 0.35, have good stereochemical qualities, contain an extensive network of hydrogen bonds consistent with the secondary structure elements and structural features in concordance with genetic data. The overall folding of the solution and crystal structures is the same.