A high-resolution solution structure of a trypanosomatid FYVE domain

FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE domain displays a remarkable specificity for the head group of the target lipid, phosphatidylinositol 3-phosphate (PtdIns[3]P). We have identified five putative FYVE domain proteins in the genome of the protozoan parasite Leishmania major, three of which are predicted to contain a functional PtdIns(3)P-binding site. The FYVE domain of one of these proteins, LmFYVE-1, bound PtdIns(3)P in liposome-binding assays and targeted GFP to acidified late endosomes/lysosomes in mammalian cells. The high-resolution solution structure of its N-terminal FYVE domain (LmFYVE-1[1-79]) was solved by nuclear magnetic resonance. Functionally significant clusters of residues of the LmFYVE-1 domain involved in PtdIns(3)P binding and dependence on low pH for tight binding were identified. This structure is the first trypanosomatid membrane trafficking protein to be determined and has been refined to high precision and accuracy using residual dipolar couplings.     

Filtering and selection of structural models: combining docking and NMR

It is generally accepted that protein structures are more conserved than protein sequences, and 3D structure determination by computer simulations have become an important necessity in the postgenomic area. Despite major successes no robust, fast, and automated ab initio prediction algorithms for deriving accurate folds of single polypeptide chains or structures of intermolecular complexes exist at present. Here we present a methodology that uses selection and filtering of structural models generated by docking of known substructures such as individual proteins or domains through easily obtainable experimental NMR constraints. In particular, residual dipolar couplings and chemical shift mapping are used. Heuristic inclusion of chemical or biochemical knowledge about point-to-point interactions is combined in our selection strategy with the NMR data and commonly used contact potentials. We demonstrate the approach for the determination of protein-protein complexes using the EIN/HPr complex as an example and for establishing the domain-domain orientation in a chimeric protein, the recently determined hybrid human-Escherichia. coli thioredoxin.     

NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.     

Application of dipolar coupling data to the refinement of the solution structure of the sarcin-ricin loop RNA

Residual dipolar couplings can provide the long-range information that most NMR solution structures lack. The use of such data in protein structure determinations is now fairly routine, but even though these data should be much more useful for nucleic acids, their application to nucleic acid structure determination is still in its infancy. Here we present a method for producing accurate, dipolar-refined structures of nucleic acids that is more efficient than those used previously, and apply it to E73, a 29 nucleotide RNA that includes the sarcin-ricin loop from rat 28S rRNA. The results enable us to address the differences between the crystal structure of E73 and the solution structure proposed for it previously.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Molecular conformations of a disaccharide investigated using NMR spectroscopy

The molecular structure of -l-Rhap-(1→ 2)--l-Rhap-OMe has been investigated using conformation sensitive NMR parameters: cross-relaxation rates, scalar ³ J _CH couplings and residual dipolar couplings obtained in a dilute liquid crystalline phase. The order matrices of the two sugar residues are different, which indicates that the molecule cannot exist in a single conformation. The conformational distribution function, , related to the two glycosidic linkage torsion angles and was constructed using the APME method, valid in the low orientational order limit. The APME approach is based on the additive potential (AP) and maximum entropy (ME) models. The analyses of the trajectories generated in molecular dynamics and Langevin dynamics (LD) computer simulations gave support to the distribution functions constructed from the experimental NMR parameters. It is shown that at least two conformational regions are populated on the Ramachandran map and that these regions exhibit very different molecular order.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Argininamide binding arrests global motions in HIV-1 TAR RNA: comparison with Mg2+-induced conformational stabilization

The structure and dynamics of the stem-loop transactivation response element (TAR) RNA from the human immunodeficiency virus type-1 (HIV-1) bound to the ligand argininamide (ARG) has been characterized using a combination of a large number of residual dipolar couplings (RDCs) and trans-hydrogen bond NMR methodology. Binding of ARG to TAR changes the average inter-helical angle between the two stems from approximately 47 degrees in the free state to approximately 11 degrees in the bound state, and leads to the arrest of large amplitude (+/-46 degrees ) inter-helical motions observed previously in the free state. While the global structural dynamics of TAR-ARG is similar to that previously reported for TAR bound to Mg2+, there are substantial differences in the hydrogen bond alignment of bulge and neighboring residues. Based on a novel H5(C5)NN experiment for probing hydrogen-mediated 2hJ(N,N) scalar couplings as well as measured RDCs, the TAR-ARG complex is stabilized by a U38-A27.U23 base-triple involving an A27.U23 reverse Hoogsteen hydrogen bond alignment as well as by a A22-U40 Watson-Crick base-pair at the junction of stem I. These hydrogen bond alignments are not observed in either the free or Mg2+ bound forms of TAR. The combined conformational analysis of TAR under three states reveals that ligands and divalent ions can stabilize similar RNA global conformations through distinct interactions involving different hydrogen bond alignments in the RNA.     

Refinement of the solution structure of the heparin-binding domain of vascular endothelial growth factor using residual dipolar couplings

Previous NMR structural studies of the heparin-binding domain of vascular endothelial growth factor (VEGF₁₆₅) revealed a novel fold comprising two subdomains, each containing two disulfide bridges and a short two-stranded antiparallel -sheet. The mutual orientation of the two subdomains was poorly defined by the NMR data. Heteronuclear relaxation data suggested that this disorder resulted from a relative lack of experimental restraints due to the limited size of the interface, rather than inherent high-frequency flexibility. Refinement of the structure using ¹H^N-¹⁵N residual dipolar coupling restraints results in significantly improved definition of the relative subdomain orientations.     

Refinement of d(GCGAAGC) hairpin structure using one- and two-bond residual dipolar couplings

The structure of the ¹³C,¹⁵N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond ¹H-¹³C, ¹H-¹⁵N, ¹³C-¹³C and ¹³C-¹⁵N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions.     

Refinement of the structure of protein–RNA complexes by residual dipolar coupling analysis

The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein–RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology.     

Residual dipolar coupling constants and structure determination of large DNA duplexes

Several NMR works have shown that long-range information provided by residual dipolar couplings (RDCs) significantly improve the global structure definition of RNAs and DNAs. Most of these are based on the use of a large set of RDCs, the collect of which requires samples labeled with ¹³C, ¹⁵N, and sometimes, ²H. Here, we carried out torsion-angle dynamics simulations on a non-self complementary DNA fragment of 17 base-pairs, d(GGAAAATATCTAGCAGT).(ACTGCTAGAGATTTTCC). This reproduces the U5 LTR distal end of the HIV-1 cDNA that contains the enzyme integrase binding site. Simulations aimed at evaluating the impact of RDCs on the structure definition of long oligonucleotides, were performed in incorporating (i) nOe-distances at both < 4.5 Å and < 5 Å; (ii) a small set of ¹³C-¹H RDCs, easily detectable at the natural abundance, and (iii) a larger set of RDCs only accessible through the ¹³C labeling of DNAs. Agreement between a target structure and a simulated structure was measured in terms of precision and accuracy. Results allowed to define conditions in which accurate DNA structures can be determined. We confirmed the strong impact of RDCs on the structure determination, and, above all, we found that a small set of RDC constraints (ca. 50) detectable at the natural abundance is sufficient to accurately derive the global and local DNA duplex structures when used in conjunction with nOe-distances < 5 Å.     

The structure of cyclolinopeptide A in chloroform refined by RDC measurements

Three‐dimensional structures of molecules traditionally assigned from nuclear Overhauser effects and vicinal coupling constants are recently complemented by measurements of residual dipolar couplings. Residual dipolar couplings measured in a stretched poly(dimethylsiloxane) gel were used to determine the structure of cyclolinopeptide A in chloroform solution at ?50 °C. After structure refinement, conformational details of main cluster were discussed in relation to crystal and nuclear Overhauser effect derived structures. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

NMR experiments for the measurement of proton-proton and carbon-carbon residual dipolar couplings in uniformly labelled oligosaccharides

A 2D-HSQC-carbon selective/proton selective-constant time COSY, 2D-HSQC-(sel C, sel H)-CT COSY experiment, which is applicable to uniformly ¹³C isotopically enriched samples (U-¹³C) of oligosaccharides or oligonucleotides is proposed for the measurement of proton–proton RDC in crowded regions of 2D-spectra. In addition, a heteronuclear constant time-COSY experiment, ¹³C-¹³C CT-COSY, is proposed for the measurement of one bond carbon–carbon RDC in these molecules. These two methods provide an extension, to U-¹³C molecules, of the original homonuclear constant time-COSY experiment proposed by Tian et al. (1999) for saccharides. The combination of a number of these RDC with NOE data may provide the method of choice to study oligosaccharide conformation in the free and receptor-bound state.     

Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G

A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is presented. MSG catalyzes the condensation of glyoxylate with an acetyl group of acetyl-CoA, producing malate, an intermediate in the citric-acid cycle. We show that despite the size of the protein, important structural and dynamic information about the molecule can be obtained on a per-residue basis. 15N-1HN residual dipolar couplings and carbonyl chemical shift changes upon alignment in Pf1 phage establish that there are no significant domain reorientations in the molecule upon ligand binding, in contrast to what was anticipated on the basis of both the X-ray structure of the glyoxylate-bound form of the enzyme and structural studies of a related set of proteins. The chemical shift changes of 1HN, 15N and 13CO nuclei upon binding of pyruvate, a glyoxylate-mimicking inhibitor, and acetyl-CoA have been mapped onto the three-dimensional structure of the molecule. Binding constants of pyruvate, glyoxylate, and acetyl-CoA (in the presence of pyruvate) have been measured, along with the kinetic parameters for glyoxylate and pyruvate binding. The on-rates of pyruvate and glyoxalate binding, approximately 1.2 x 10(6)M(-1)s(-1) and approximately 2.7 x 10(6)M(-1)s(-1), respectively, are significantly lower than what is anticipated from a simple diffusion-controlled process. Some structural implications of the chemical shift perturbations upon binding and the estimated ligand on-rates are discussed.     

Structure determination of a new protein from backbone-centered NMR data and NMR-assisted structure prediction

Targeting of proteins for structure determination in structural genomic programs often includes the use of threading and fold recognition methods to exclude proteins belonging to well-populated fold families, but such methods can still fail to recognize preexisting folds. The authors illustrate here a method in which limited amounts of structural data are used to improve an initial homology search and the data are subsequently used to produce a structure by data-constrained refinement of an identified structural template. The data used are primarily NMR-based residual dipolar couplings, but they also include additional chemical shift and backbone-nuclear Overhauser effect data. Using this methodology, a backbone structure was efficiently produced for a 10 kDa protein (PF1455) from Pyrococcus furiosus. Its relationship to existing structures and its probable function are discussed.     

NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperone

Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.     

Assignment-free solution NMR method reveals CesT as an unswapped homodimer

The X-ray structure of the homodimeric chaperone CesT is the only structure among the type three secretion system (TTSS) chaperones that shows a domain swap. This swap has potential importance for the mechanism of effector translocation through a TTSS. Here we present two nuclear magnetic resonance strategies exploiting pre-existing structural models and residual dipolar couplings (RDCs), which reveal the unswapped 35.4-kDa dimer to be present in solution. Particularly efficient is the discrimination of a swapped and unswapped structural state performed simultaneously to automatic backbone assignment using only HN-RDCs and carbonyl backbone chemical shifts. This direct approach may prove to be generally useful to rapidly differentiate two structural models.     

Solution structure of the GUCT domain from human RNA helicase II/Gu beta reveals the RRM fold, but implausible RNA interactions

Human RNA helicase II/Gu alpha (RH-II/Gu alpha) and RNA helicase II/Gu beta (RH-II/Gu beta) are paralogues that share the same domain structure, consisting of the DEAD box helicase domain (DEAD), the helicase conserved C-terminal domain (helicase_C), and the GUCT domain. The N-terminal regions of the RH-II/Gu proteins, including the DEAD domain and the helicase_C domain, unwind double-stranded RNAs. The C-terminal tail of RH-II/Gu alpha, which follows the GUCT domain, folds a single RNA strand, while that of RH-II/Gu beta does not, and the GUCT domain is not essential for either the RNA helicase or foldase activity. Thus, little is known about the GUCT domain. In this study, we have determined the solution structure of the RH-II/Gu beta GUCT domain. Structural calculations using NOE-based distance restraints and residual dipolar coupling-based angular restraints yielded a well-defined structure with beta-alpha-alpha-beta-beta-alpha-beta topology in the region for K585-A659, while the Pfam HMM algorithm defined the GUCT domain as G571-E666. This structure-based domain boundary revealed false positives in the sequence homologue search using the HMM definition. A structural homology search revealed that the GUCT domain has the RRM fold, which is typically found in RNA-interacting proteins. However, it lacks the surface-exposed aromatic residues and basic residues on the beta-sheet that are important for the RNA recognition in the canonical RRM domains. In addition, the overall surface of the GUCT domain is fairly acidic, and thus the GUCT domain is unlikely to interact with RNA molecules. Instead, it may interact with proteins via its hydrophobic surface around the surface-exposed tryptophan.