Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3

Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free-state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin-3, and report enhanced RDCs that prove consistent with known bound-ligand geometries for this protein.     

Three-dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

The traditional NMR‐based method for determining oligomeric protein structure usually involves distinguishing and assigning intra‐ and intersubunit NOEs. This task becomes challenging when determining symmetric homo‐dimer structures because NOE cross‐peaks from a given pair of protons occur at the same position whether intra‐ or intersubunit in origin. While there are isotope‐filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K_d 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape‐based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.     

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand–protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand–protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands.     

15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c

Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz. The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints. Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein. By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein. The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states. The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility. Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.     

Structural characterization of a mannose-binding protein-trimannoside complex using residual dipolar couplings

The ligand-binding properties of a 53 kDa homomultimeric trimer from mannose-binding protein (MBP) have been investigated using residual dipolar couplings (RDCs) that are easily measured from NMR spectra of the ligand and isotopically labeled protein. Using a limited set of 1H-15N backbone amide NMR assignments for MBP and orientational information derived from the RDC measurements in aligned media, an order tensor for MBP has been determined that is consistent with symmetry-based predictions of an axially symmetric system. 13C-1H couplings for a bound trisaccharide ligand, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (trimannoside) have been determined at natural abundance and used as orientational constraints. The bound ligand geometry and orientational constraints allowed docking of the trimannoside ligand in the binding site of MBP to produce a structural model for MBP-oligosaccharide interactions.     

Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings

Residual dipolar couplings for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This approach is used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin. Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (+/-4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide.     

Global orientation of bound MMP-3 and N-TIMP-1 in solution via residual dipolar couplings

Crystal structures of catalytic domains of MMP-3 and MT1-MMP bound to TIMP-1 or TIMP-2, respectively, differ in the orientation of the TIMP in the MMP active site. The orientation in solution of N-TIMP-1 in the MMP-3 active site has been investigated using residual dipolar couplings (RDCs). Fitting of the RDCs to the X-ray structures of the complexes suggests general agreement with the orientation of crystalline MMP-3(DeltaC) and TIMP-1 and a large disparity from the orientation of crystalline MT1-MMP(DeltaC) and TIMP-2. Rigid body docking of MMP-3 and N-TIMP-1 X-ray coordinates using RDCs and intermolecular NOEs provided a time-averaged orientation in solution differing from the crystal structure by a 5 degrees rotation toward the MT1-MMP(DeltaC)/TIMP-2 orientation. The slight discrepancy in orientations in solution and crystal lies within the experimental uncertainties. Intermolecular NOEs used in the docking corroborated the accuracy of mapping the interface by a paramagnetic NMR footprinting assay, a potential alternative source of contacts for docking. Some uncertainty in the N-TIMP-1 orientation in the MMP-3 active site, coupled with microsecond to millisecond fluctuations of the MMP-binding ridge of N-TIMP-1 in the complex and flexibility in MMP-3(DeltaC) S(1)' to S(3)' subsites, leaves open the possibility that N-TIMP-1 might dynamically pivot a few degrees or more in the arc toward the MT1-MMP(DeltaC)/TIMP-2 orientation. Differing TIMP orientations in MMP active sites are more likely to result from structural differences in TIMP AB hairpin loops than from crystal packing artifacts.     

Fast methionine-based solution structure determination of calcium-calmodulin complexes

Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca²⁺-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaM’s backbone conformation and a structural plasticity in CaM’s domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant. Although RDCs can define CaM domain orientation in the complex, they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs. Here we employ a specific isotope labeling strategy in which the role of methionine in CaM-peptide interactions is exploited to collect these critical NOEs. By ¹H, ¹³C-labeling the methyl groups of deuterated methionine against a ²H, ¹²C background, we can acquire a ¹³C-edited NOESY characterized by simplified, easily analyzable spectra. Together with measured CaM backbone H_N-N RDCs and intrapeptide NOE-based distances, these intermolecular NOEs provide restraints for a low temperature torsion-angle dynamics and simulated annealing protocol used to calculate the complex structure. We have applied our method to a CaM complex previously solved through X-ray crystallography: Ca²⁺-CaM bound to the CaM kinase I peptide (PDB code: 1MXE). The resulting structure has a backbone RMSD of 1.6 Å to that previously published. We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome. In addition to having application for fast complex structure determination, this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully ¹³C, ¹⁵N-labeled CaM fails.     

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein–ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.     

Identification of individual protein–ligand NOEs in the limit of intermediate exchange

Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations. Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we employ methods related to driven NOE spectroscopy to investigate protein–ligand interactions in the intermediate exchange regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples selectively protonated at a few sites so that the 1D ¹H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein–ligand NOEs that allow calculation of initial complex structures, suitable for structure-based optimization of primary drug leads obtained from high-throughput screening. The method was applied to measure individual intermolecular NOEs between the anti-apoptotic protein Bcl-xL at 25 μM and a “first generation” small-molecule ligand, for which the spectrum is entirely broadened at stoichiometric concentrations. This approach is general and can also be used to characterize protein–protein or protein–nucleic-acid complexes.     

Structural and Functional Mimicry of the Binding Site of hYAP-WW Domain for Proline-rich Ligands

Based on the 3D structure of the WW domain of human Yes-associated protein (hYAP-WW) in complex with a proline-rich peptide ligand, we have designed and synthesized a cyclic peptide covering a fragment of hYAP-WW that contains its primary contact residues for the interaction with the ligand. This peptide was found to specifically recognize a proline-rich ligand for hYAP-WW. Its conformation was calculated using molecular dynamics simulation, based on long-range NOEs identified by NMR spectroscopy, and indicates an arrangement of primary contact residues similar to hYAP-WW.     

Methyl group reorientation under ligand binding probed by pseudocontact shifts

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1–3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.     

Determination of solution structures of paramagnetic proteins by NMR

Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron-nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints from NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8. Received: 13 October 1997 / Accepted: 19 December 1997     

Accuracy of bound peptide structures determined by exchange transferred nuclear Overhauser data: A simulation study

The exchange-transferred NOE method to determine the three-dimensional structure of peptides bound to proteins, or other macromolecular systems, is becoming increasingly important in drug design efforts and for large or multicomponent assemblies, such as membrane receptors, where structural analysis of the full system is intractable. The exchange-transferred nuclear Overhauser effect spectroscopy (etNOESY) method allows the determination of the bound-state conformation of the peptide from the intra-molecular NOE interactions between ligand protons. Because only ligand–ligand NOEs are generally observable, the etNOESY method is restricted to fewer NOEs per residue than direct protein structure determination. In addition, the averaging of relaxation rates between free and bound states affects the measured cross-peak intensities, and possibly the accuracy of distance estimates. Accordingly, the study reported here was conducted to examine the conditions required to define a reliable structure. The program CORONA was used to simulate etNOE data using a rate-matrix including magnetic relaxation and exchange rates for two peptide–protein complexes derived from the reference complex of cAMP-dependent protein kinase ligated with a 24-residue inhibitor peptide. The results indicate that reasonably accurate peptide structures can be determined with relatively few NOE interactions when the interactions occur between non-neighboring residues. The reliability of the structural result is suggested from the pattern of NOE interactions. A structure with an accuracy of approximately 1.3 Å rms difference for the main-chain atoms can be obtained when etNOE interactions between non-neighboring residues occur over the length of the peptide. The global precision is higher (approximately 0.9 Å rms difference) but is not correlated to global accuracy. A local definition of precision along the backbone appears to be a good indicator of the local accuracy.     

Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments

Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.     

Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts

Pseudocontact shift (PCS) effects induced by a paramagnetic lanthanide bound to a protein have become increasingly popular in NMR spectroscopy as they yield a complementary set of orientational and long-range structural restraints. PCS are a manifestation of the chi-tensor anisotropy, the Deltachi-tensor, which in turn can be determined from the PCS. Once the Deltachi-tensor has been determined, PCS become powerful long-range restraints for the study of protein structure and protein-ligand complexes. Here we present the newly developed package Numbat (New User-friendly Method Built for Automatic Deltachi-Tensor determination). With a Graphical User Interface (GUI) that allows a high degree of interactivity, Numbat is specifically designed for the computation of the complete set of Deltachi-tensor parameters (including shape, location and orientation with respect to the protein) from a set of experimentally measured PCS and the protein structure coordinates. Use of the program for Linux and Windows operating systems is illustrated by building a model of the complex between the E. coli DNA polymerase III subunits epsilon186 and theta using PCS.     

Capturing Conformational States in Proteins Using Sparse Paramagnetic NMR Data

Capturing conformational changes in proteins or protein-protein complexes is a challenge for both experimentalists and computational biologists. Solution nuclear magnetic resonance (NMR) is unique in that it permits structural studies of proteins under greatly varying conditions, and thus allows us to monitor induced structural changes. Paramagnetic effects are increasingly used to study protein structures as they give ready access to rich structural information of orientation and long-range distance restraints from the NMR signals of backbone amides, and reliable methods have become available to tag proteins with paramagnetic metal ions site-specifically and at multiple sites. In this study, we show how sparse pseudocontact shift (PCS) data can be used to computationally model conformational states in a protein system, by first identifying core structural elements that are not affected by the environmental change, and then computationally completing the remaining structure based on experimental restraints from PCS. The approach is demonstrated on a 27 kDa two-domain NS2B-NS3 protease system of the dengue virus serotype 2, for which distinct closed and open conformational states have been observed in crystal structures. By changing the input PCS data, the observed conformational states in the dengue virus protease are reproduced without modifying the computational procedure. This data driven Rosetta protocol enables identification of conformational states of a protein system, which are otherwise difficult to obtain either experimentally or computationally.     

Structure and dynamics in solution of the complex of Lactobacillus casei dihydrofolate reductase with the new lipophilic antifolate drug trimetrexate

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate. Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N-labeled protein. Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints. Several intermolecular protein-ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand. At low temperature (5 degrees C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks. At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower-temperature spectrum to be identified as NOEs involving ligand protons. The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conformational differences were detected between the different complexes. The 2,4-diaminopyrimidine-containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side-chain orientations and local conformations of which are not greatly changed in the different complexes (similar chi1 and chi2 values).     

Structural investigations of a GYF domain covalently linked to a proline-rich peptide

Protein structure determination of low affinity complexes of interacting macromolecules is often hampered by a lack of observable NOEs between the binding partners. Covalent linkage offers a way to shift the equilibrium of the interaction partners to the bound state. Here we show that a single-chain protein containing the GYF domain of CD2BP2 and the target peptide SHRPPPPGHRV from CD2 allows for the intramolecular association of the binding partners. We obtained NOEs between the GYF domain and the peptide that could define the principal orientation of the peptide in the complex. In conjunction with general recognition rules for proline-rich sequence recognition these NOEs allowed the accurate modeling of the protein-peptide complex.     

An improved algorithm for MFR fragment assembly

A method for generating protein backbone models from backbone only NMR data is presented, which is based on molecular fragment replacement (MFR). In a first step, the PDB database is mined for homologous peptide fragments using experimental backbone-only data i.e. backbone chemical shifts (CS) and residual dipolar couplings (RDC). Second, this fragment library is refined against the experimental restraints. Finally, the fragments are assembled into a protein backbone fold using a rigid body docking algorithm using the RDCs as restraints. For improved performance, backbone nuclear Overhauser effects (NOEs) may be included at that stage. Compared to previous implementations of MFR-derived structure determination protocols this model-building algorithm offers improved stability and reliability. Furthermore, relative to CS-ROSETTA based methods, it provides faster performance and straightforward implementation with the option to easily include further types of restraints and additional energy terms.