Paramagnetic Restraints Research Articles

Characterization of lanthanoid-binding proteins using NMR spectroscopy.

The variety of magnetic properties exhibited by paramagnetic lanthanoids provides outstanding information in NMR-based structural biology and therefore can be a very useful tool for characterizing lanthanoid-binding proteins. Because of their dependence on the relative positions of the protein nuclei and of the lanthanoid ion, the paramagnetic restraints (PCS, PRDC and PRE) provide information on structure and dynamics of proteins. In this Chapter, we cover the use of lanthanoids in structural biology including protein sample preparation, NMR experiments and data interpretation.

Paramagnetic NMR in drug discovery

The presence of an unpaired electron in paramagnetic molecules generates significant effects in NMR spectra, which can be exploited to provide restraints complementary to those used in standard structure-calculation protocols. NMR already occupies a central position in drug discovery for its use in fragment screening, structural biology and validation of ligand–target interactions. Paramagnetic restraints provide unique opportunities, for example, for more sensitive screening to identify weaker-binding fragments. A key application of paramagnetic NMR in drug discovery, however, is to provide new structural restraints in cases where crystallography proves intractable. This is particularly important at early stages in drug-discovery programs where crystal structures of weakly-binding fragments are difficult to obtain and crystallization artefacts are probable, but structural information about ligand poses is crucial to guide medicinal chemistry. Numerous applications show the value of paramagnetic restraints to filter computational docking poses and to generate interaction models. Paramagnetic relaxation enhancements (PREs) generate a distance-dependent effect, while pseudo-contact shift (PCS) restraints provide both distance and angular information. Here, we review strategies for introducing paramagnetic centers and discuss examples that illustrate the utility of paramagnetic restraints in drug discovery. Combined with standard approaches, such as chemical shift perturbation and NOE-derived distance information, paramagnetic NMR promises a valuable source of information for many challenging drug-discovery programs.

The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins.

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein‐ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide‐binding‐tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo‐catalyzed thiol‐ene reaction for the cysteine‐selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl‐pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.

Integrative Protein Modeling in RosettaNMR from Sparse Paramagnetic Restraints

Computational methods to predict protein structure from nuclear magnetic resonance (NMR) restraints that only require assignment of backbone signals, hold great potential to study larger proteins. Ideally, computational methods designed to work with sparse data need to add atomic detail that is missing in the experimental restraints. We introduce a comprehensive framework into the Rosetta suite that uses NMR restraints derived from paramagnetic labeling. Specifically, RosettaNMR incorporates pseudocontact shifts, residual dipolar couplings, and paramagnetic relaxation enhancements. It continues to use backbone chemical shifts and nuclear Overhauser effect distance restraints. We assess RosettaNMR for protein structure prediction by folding 28 monomeric proteins and 8 homo-oligomeric proteins. Furthermore, the general applicability of RosettaNMR is demonstrated on two protein-protein and three protein-ligand docking examples. Paramagnetic restraints generated more accurate models for 85% of the benchmark proteins and, when combined with chemical shifts, sampled high-accuracy models (≤2Å) in 50% of the cases.

Active-Site Targeting Paramagnetic Probe for Matrix Metalloproteinases.

The design and synthesis of the Ln3+ complexes of a DOTA-containing (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) inhibitor of matrix metalloproteinases are reported. The tight binding of the sulfonamide scaffold to the catalytic domain of the investigated matrix metalloproteinase is not impaired by the presence of the Ln3+ -DOTA moiety. The paramagnetic properties of the Ln3+ complex are exploited to obtain insights into the structural features of the ligand-protein interactions and to evaluate the influence of the linker length on the quality of the paramagnetic restraints.

Ensemble structure description of Lys63-linked diubiquitin.

The data described herein are related to the article entitled “Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition” [1], and to the coordinates for the ensemble structure of Lys63-linked diubiquitin (PDB code 2N2K). A Lys63-linked diubiquitin exists in three conformational states with different orientations for the two subunits, each responsible for binding to a target protein and encoding a specific cell signal. An atomic entry in the ensemble structure file consists multiple lines, representing alternative locations of the atom and recapitulating the dynamics of the protein. Experimental details about obtaining strictly intramolecular paramagnetic restraints and determining the relative occupancies of the conformational states are presented. The experimental design and procedures in this Data article can be useful for characterizing the structure and dynamics of other multi-domain proteins.

Multiple paramagnetic effects through a tagged reporter protein.

Paramagnetic effects provide unique information about the structure and dynamics of biomolecules. We developed a method in which the lanthanoid tag is not directly attached to the protein of interest, but instead to a "reporter" protein, which binds and then transmits paramagnetic information to the target. The designed method allows access to a large number of paramagnetic restraints and residual dipolar couplings produced from independent molecular alignments in high-molecular-weight proteins with unknown 3D structure.

Structure determination of protein–protein complexes with long-range anisotropic paramagnetic NMR restraints

Paramagnetic NMR spectroscopy has evolved rapidly in the last decade, and has shown to be a very useful tool for solving structures of protein-protein complexes. A major breakthrough has been the development of paramagnetic metal binding tags that can be attached specifically to the protein. These tags have greatly facilitated the use of anisotropic paramagnetic restraints such as pseudocontact shifts and residual dipolar couplings arising from paramagnetic self-alignment. Such restraints are particularly useful for the study of large protein complexes. This review focuses on the recent developments in structural characterization of protein-protein complexes using anisotropic paramagnetic NMR restraints.

Bioconjugation of Proteins with a Paramagnetic NMR and Fluorescent Tag

Site-specific labeling of proteins with lanthanide ions offers great opportunities for investigating the structure, function, and dynamics of proteins by virtue of the unique properties of lanthanides. Lanthanide-tagged proteins can be studied by NMR, X-ray, fluorescence, and EPR spectroscopy. However, the rigidity of a lanthanide tag in labeling of proteins plays a key role in the determination of protein structures and interactions. Pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) are valuable long-range structure restraints in structural-biology NMR spectroscopy. Generation of these paramagnetic restraints generally relies on site-specific tagging of the target proteins with paramagnetic species. To avoid nonspecific interaction between the target protein and paramagnetic tag and achieve reliable paramagnetic effects, the rigidity, stability, and size of lanthanide tag is highly important in paramagnetic labeling of proteins. Here 4'-mercapto-2,2':6',2''-terpyridine-6,6''-dicarboxylic acid (4MTDA) is introduced as a a rigid paramagnetic and fluorescent tag which can be site-specifically attached to a protein by formation of a disulfide bond. 4MTDA can be readily immobilized by coordination of the protein side chain to the lanthanide ion. Large PCSs and RDCs were observed for 4MTDA-tagged proteins in complexes with paramagnetic lanthanide ions. At an excitation wavelength of 340 nm, the complex formed by protein-4MTDA and Tb(3+) produces high fluorescence with the main emission at 545 nm. These interesting features of 4MTDA make it a very promising tag that can be exploited in NMR, fluorescence, and EPR spectroscopic studies on protein structure, interaction, and dynamics.

Solid-State NMR Crystallography through Paramagnetic Restraints

Pseudocontact shifts (PCSs) measured by solid-state NMR spectroscopy (SS-NMR) on microcrystalline powders of a paramagnetic metalloprotein permit NMR crystallography. Along with other restraints for SS-NMR experiments, the protein molecular structure as well as the correct crystal packing are obtained.

Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains

Calmodulin is a two-domain protein which in solution can adopt a variety of conformations upon reorientation of its domains. The maximum occurrence (MO) of a set of calmodulin conformations that are representative of the overall conformational space possibly sampled by the protein, has been calculated from the paramagnetism-based restraints. These restraints were measured after inclusion of a lanthanide binding tag in the C-terminal domain to supplement the data obtained by substitution of three paramagnetic lanthanide ions to the calcium ion in the second calcium binding loop of the N-terminal domain. The analysis shows that the availability of paramagnetic restraints arising from metal ions placed on both domains, reduces the MO of the conformations to different extents, thereby helping to identify those conformations that can be mostly sampled by the protein.

Engineering encodable lanthanide-binding tags into loop regions of proteins.

Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (IL1β) and varied the length of the spacer between the LBT and the protein (denoted 1−3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing the 1H−15N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling.

A Solution Model of the Complex Formed by Adrenodoxin and Adrenodoxin Reductase Determined by Paramagnetic NMR Spectroscopy

Lanthanide tags offer the opportunity to retrieve long-range distance information from NMR experiments that can be used to guide protein docking. To determine whether sufficient restraints can be retrieved for proteins with low solubility and availability, Ln tags were applied in the study of the 65 kDa membrane-associated protein complex formed by the electron carrier adrenodoxin and its electron donor, adrenodoxin reductase. The reductase is only monomeric at low concentration, and the paramagnetic iron-sulfur cluster of adrenodoxin broadens many of the resonances of nuclei in the interface. Guided by the paramagnetic restraints obtained using two Ln-tag attachment sites, protein docking yields a cluster of solutions with an rmsd of 3.2 A. The mean structure is close to the crystal structure of the cross-linked complex, with an rmsd of 4.0 A. It is concluded that with the application of Ln tags paramagnetic NMR restraints for structure determination can be retrieved even for difficult, low-concentration protein complexes.

Accurate Solution Structures of Proteins from X-ray Data and a Minimal Set of NMR Data: Calmodulin−Peptide Complexes As Examples

A strategy for the accurate determination of protein solution structures starting from X-ray data and a minimal set of NMR data is proposed and successfully applied to two complexes of calmodulin (CaM) with target peptides not previously described. Its implementation in the present case is based on the use of lanthanide ions as substitutes for calcium in one of the four calcium binding sites of CaM and the collection of pseudocontact shift (pcs) and residual dipolar coupling (rdc) restraints induced by the paramagnetic metals. Starting from the crystal structures, new structural models are calculated that are in excellent agreement with the paramagnetic restraints and differ significantly from the starting crystal structures. In particular, in both complexes, a change in orientation of the first helix of the N-terminal CaM domain and of the whole C-terminal domain is observed. The simultaneous use of paramagnetic pcs and rdc restraints has the following crucial advantages: (i) it allows one to assess the possible presence of interdomain conformational freedom, which cannot be detected if the rdc values are derived from external orienting media; (ii) in the absence of significant conformational freedom, the global orientation tensor can be independently and precisely determined from pcs values, which are less sensitive than rdc values to the presence of local structural inaccuracies, and therefore (iii) the relative rearrangement of a domain or a secondary structure element with respect to the metal-bearing domain can be detected.

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR.

The biological function of metalloproteins is closely tied to the geometric and electronic structures of the metal sites. Here, we show that the geometric structure of the metal site of a metalloprotein in solution can be determined from experimentally measured electron-nuclear spin-spin interactions obtained by NMR. Thus, the geometric metal site structure of plastocyanin from Anabaena variabilis was determined by including the paramagnetic relaxation enhancement of protons close to the copper site as restraints in a conventional NMR structure determination, together with the distribution of the unpaired electron onto the ligand atoms. Also, the interproton distances (nuclear Overhauser enhancements) and dihedral angles (scalar nuclear spin-spin couplings) normally used in NMR structure determinations were included as restraints. The structure calculations were carried out with the program X-PLOR and a module that takes into account the specific characteristics of the paramagnetic restraints. A well defined metal site structure was obtained with the structural characteristics of the blue copper site, including a distorted tetrahedral geometry, a short Cu-Cys S gamma bond, and a long Cu-Met S delta bond. Overall, the agreement of the obtained metal site structure of Anabaena variabilis plastocyanin with those of other plastocyanins obtained by x-ray crystallography confirms the reliability of the approach.

Investigation of the binding geometry of a peripheral membrane protein.

A growing number of modules including FYVE domains target key signaling proteins to membranes through specific recognition of lipid headgroups and hydrophobic insertion into bilayers. Despite the critical role of membrane insertion in the function of these modules, the structural mechanism of membrane docking and penetration remains unclear. In particular, the three-dimensional orientation of the inserted proteins with respect to the membrane surface is difficult to define quantitatively. Here, we determined the geometry of the micelle penetration of the early endosome antigen 1 (EEA1) FYVE domain by obtaining NMR-derived restraints that correlate with the distances between protein backbone amides and spin-labeled probes. The 5- and 14-doxyl-phosphatidylcholine spin-labels were incorporated into dodecylphosphocholine (DPC) micelles, and the reduction of amide signal intensities of the FYVE domain due to paramagnetic relaxation enhancement was measured. The vector of the FYVE domain insertion was estimated relative to the molecular axis by minimizing the paramagnetic restraints obtained in phosphatidylinositol 3-phosphate (PI3P)-enriched micelles containing only DPC or mixed with phosphatidylserine (PS). Additional distance restraints were obtained using a novel spin-label mimetic of PI(3)P that contains a nitroxyl radical near the threitol group of the lipid. Conformational changes indicative of elongation of the membrane insertion loop (MIL) were detected upon micelle interaction, in which the hydrophobic residues of the loop tend to move deeper into the nonpolar core of micelles. The micelle insertion mechanism of the FYVE domain defined in this study is consistent with mutagenesis data and chemical shift perturbations and demonstrates the advantage of using the spin-label NMR approach for investigating the binding geometry by peripheral membrane proteins.

Site-specific labelling with a metal chelator for protein-structure refinement.

A single free Cys sidechain in the N-terminal domain of the E. coli arginine repressor was covalently derivatized with S-cysteaminyl-EDTA for site-specific attachment of paramagnetic metal ions. The effects of chelated metal ions were monitored with (15)N-HSQC spectra. Complexation of Co(2+), which has a fast relaxing electron spin, resulted in significant pseudocontact shifts, but also in peak doubling which was attributed to the possibility of forming two different stereoisomers of the EDTA-Co(2+) complex. In contrast, complexation of Cu(2+) or Mn(2+), which have slowly relaxing electron spins, did not produce chemical shift changes and yielded self-consistent sets of paramagnetic relaxation enhancements of the amide protons. T (1) relaxation enhancements with Cu(2+) combined with T (2) relaxation enhancements with Mn(2+) are shown to provide accurate distance restraints ranging from 9 to 25 A. These long-range distance restraints can be used for structural studies inaccessible to NOEs. As an example, the structure of a solvent-exposed loop in the N-terminal domain of the E. coli arginine repressor was refined by paramagnetic restraints. Electronic correlation times of Cu(2+) and Mn(2+) were determined from a comparison of T (1) and T (2) relaxation enhancements.