Multidimensional Heteronuclear NMR Research Articles

Structural Details on mdm2-p53 Interaction

Mdm2 is a cellular antagonist of p53 that keeps a balanced cellular level of p53. The two proteins are linked by a negative regulatory feedback loop and physically bind to each other via a putative helix formed by residues 18-26 of p53 transactivation domain (TAD) and its binding pocket located within the N-terminal 100-residue domain of mdm2 (Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996) Science 274, 948-953). In a previous report we demonstrated that p53 TAD in the mdm2-freee state is mostly unstructured but contains two nascent turns in addition to a "preformed" helix that is the same as the putative helix mediating p53-mdm2 binding. Here, using heteronuclear multidimensional NMR methods, we show that the two nascent turn motifs in p53 TAD, turn I (residues 40-45) and turn II (residues 49-54), are also capable of binding to mdm2. In particular, the turn II motif has a higher mdm2 binding affinity ( approximately 20 mum) than the turn I and targets the same site in mdm2 as the helix. Upon mdm2 binding this motif becomes a well defined full helix turn whose hydrophobic face formed by the side chains of Ile-50, Trp-53, and Phe-54 inserts deeply into the helix binding pocket. Our results suggest that p53-mdm2 binding is subtler than previously thought and involves global contacts such as multiple "non-contiguous" minimally structured motifs instead of being localized to one small helix mini-domain in p53 TAD.

Structural and Genetic Analyses Reveal a Key Role in Prophage Excision for the TorI Response Regulator Inhibitor

TorI (Tor inhibition protein) has been identified in Escherichia coli as a protein inhibitor acting through protein-protein interaction with the TorR response regulator. This interaction, which does not interfere with TorR DNA binding activity, probably prevents the recruitment of RNA polymerase to the torC promoter. In this study we have solved the solution structure of TorI, which adopts a prokaryotic winged-helix arrangement. Despite no primary sequence similarity, the three-dimensional structure of TorI is highly homologous to the (lambda)Xis, Mu bacteriophage repressor (MuR-DBD), and transposase (MuA-DBD) structures. We propose that the TorI protein is the structural missing link between the (lambda)Xis and MuR proteins. Moreover, in vivo assays demonstrated that TorI plays an essential role in prophage excision. Heteronuclear NMR experiments and site-directed mutagenesis studies have pinpointed out key residues involved in the DNA binding activity of TorI. Our findings suggest that TorI-related proteins identified in various pathogenic bacterial genomes define a new family of atypical excisionases.

NMR Methods for Studying the Structure and Dynamics of RNA

Proper functioning of RNAs requires the formation of complex three-dimensional structures combined with the ability to rapidly interconvert between multiple functional states. This review covers recent advances in isotope-labeling strategies and NMR experimental approaches that have promise for facilitating solution structure determinations and dynamics studies of biologically active RNAs. Improved methods for the production of isotopically labeled RNAs combined with new multidimensional heteronuclear NMR experiments make it possible to dramatically reduce spectral crowding and simplify resonance assignments for RNAs. Several novel applications of experiments that directly detect hydrogen-bonding interactions are discussed. These studies demonstrate how NMR spectroscopy can be used to distinguish between possible secondary structures and identify mechanisms of ligand binding in RNAs. A variety of recently developed methods for measuring base and sugar residual dipolar couplings are described. NMR residual dipolar coupling techniques provide valuable data for determining the long-range structure and orientation of helical regions in RNAs. A number of studies are also presented where residual dipolar coupling constraints are used to determine the global structure and dynamics of RNAs. NMR relaxation data can be used to probe the dynamics of macromolecules in solution. The power dependence of transverse rotating-frame relaxation rates was used here to study dynamics in the minimal hammerhead ribozyme. Improved methods for isotopically labeling RNAs combined with new types of structural data obtained from a growing repertoire of NMR experiments are facilitating structural and dynamic studies of larger RNAs.

Heteronuclear Solution-State NMR Studies of the Chromophore in Cyanobacterial Phytochrome Cph1

Precise structural information regarding the chromophore binding pocket is essential for an understanding of photochromicity and photoconversion in phytochrome photoreceptors. To this end, we are studying the 59 kDa N-terminal module of the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 in both thermally stable forms (Pr and Pfr) using solution-state NMR spectroscopy. The protein is deuterated, while the chromophore, phycocyanobilin (PCB), is isotopically labeled with (15)N or (13)C and (15)N. We have established a simple approach for preparing labeled PCB based on BG11 medium supplemented with an appropriate buffer and NaH(13)CO(3) and Na(15)NO(3) as sole carbon and nitrogen sources, respectively. We show that structural details of the chromophore binding pocket in both Pr and Pfr forms can be obtained using multidimensional heteronuclear solution-state NMR spectroscopy. Using one-dimensional (15)N NMR spectra, we show unequivocally that the chromophore is protonated in both Pr and Pfr states.

The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes

Resuscitation-promoting factor (RPF) proteins reactivate stationary-phase cultures of (G+C)-rich Gram-positive bacteria including the causative agent of tuberculosis, Mycobacterium tuberculosis. We report the solution structure of the RPF domain from M. tuberculosis Rv1009 (RpfB) solved by heteronuclear multidimensional NMR. Structural homology with various glycoside hydrolases suggested that RpfB cleaved oligosaccharides. Biochemical studies indicate that a conserved active site glutamate is important for resuscitation activity. These data, as well as the presence of a clear binding pocket for a large molecule, indicate that oligosaccharide cleavage is probably the signal for revival from dormancy.

Cis/trans heterogeneity of Gln30-Pro31 peptide bond determines whether a 79-residue fragment of staphylococcal nuclease self-associates

The self-association reaction of a 79-residue fragment of staphylococcal nuclease (SNase79) was studied by far-UV CD, size-exclusion chromatography, and heteronuclear multidimensional NMR spectroscopy. A large population of SNase79 is in self-associated state while a small population of SNase79 is essentially in a monomeric state. The sequence region Thr13-Val39 is responsible for association interface of SNase79. The trans-conformation of X-prolyl bond Gln30-Pro31 may make residues Tyr27-Gln30, serve as a folding nucleation site, and lead the segment Thr13-Val39 of SNase79 to adopt a native-like β-sheet conformation, which results in the self-association of SNase79. The non-native conformation of the segment Thr13-Val39 of SNase79 associated with the cis-conformation of X-prolyl bond Gln30-Pro31 may preclude SNase79 from the soluble aggregates.

NMR structure of HI0004, a putative essential gene product from Haemophilus influenzae, and comparison with the X‐ray structure of an Aquifex aeolicus homolog

The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a alpha-beta-alpha sandwich architecture consisting of a central four-stranded beta-sheet with the alpha2-helix packed against one side of the beta-sheet and four alpha-helices (alpha1, alpha3, alpha4, alpha5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X-ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.

When Monomers Are Preferred: A Strategy for the Identification and Disruption of Weakly Oligomerized Proteins

Oligomerization is important for the structure and function of many proteins, but frequently complicates their characterization. It is often desirable to obtain the protein in monomeric form. Here, we report a strategy that allows the generation of monomers from weakly associated oligomers but does not require knowledge of the three-dimensional structure of the protein. The dynamics of protein association are used in solution NMR spectroscopy to identify regions of the polypeptide chain that are likely to be responsible for the interaction. Protein sequence analysis further refines the selection, as conserved sites with moderate hydrophobicity are targeted for modification. Gel filtration and activity assays straightforwardly reveal the consequences of the change and are used to screen for the desired mutants. The strategy is demonstrated for the Rac1 binding domain of plexin-B1. A monomeric variant is generated which preserves the Rac1 binding activity and the wild-type protein structure.

NMR Structural Analysis of Factor XI Apple 4 Domain.

Blood coagulation is achieved by two closely coordinated mechanisms: i) the contact factor, or intrinsic, pathway initiated by assembly of coagulation proteins on negatively charged surfaces, and ii) the extrinsic pathway initiated by exposure of tissue factor at the site of vascular injury. Although factor XI (FXI), a 160 KDa homodimeric plasma coagulation protein, can be activated either by FXIIa (generated by the contact factor pathway) or by thrombin (generated by the extrinsic or tissue factor pathway), recent evidence suggests that its unique dimeric structure is required for FXI-activation by thrombin on the platelet surface, leading to the initiation of the intrinsic pathway that is required for normal hemostasis. Each FXI monomer consists of an N-terminal heavy chain and a C-terminal trypsin-like catalytic light chain. The heavy chain consists of four homologous subunits called apple domains (designated A1 to A4). The A2 and A3 domains of one monomeric subunit bind to FIX, whereas the A3 domain of the other monomeric subunit binds to platelets. The A4 domain, which shares high (25–38%) sequence identity with other Apple domains, facilitates FXI dimer formation through an intermolecular disulfide bond at Cys-321. In the present study the rA4 domain was cloned and purified to determine its three-dimensional structure. Multidimensional heteronuclear NMR experiments were carried out using C13, N15, H2 labeled samples. Chemical shifts of the C13, N15 and H1 resonance of all the residues were assigned. Assignment of NOE cross peaks between inter- and intra-subunit amino acids is in progress. Preliminary results indicate that the monomeric structure of the A4 domain consists of six anti-parallel β-strands and an α-helix, stabilized by three cystine cross links. The orientations of charged residues and hydrophobic patches on different sides of the molecule may play important roles in the dimerization process of FXI.

Mapping the Binding of the N-terminal Extracellular Tail of the CXCR4 Receptor to Stromal Cell-derived Factor-1α

The solution structure of monomeric stromal cell-derived factor-1alpha (SDF-1alpha), the natural ligand for the CXCR4 G-coupled receptor, has been solved by multidimensional heteronuclear NMR spectroscopy. The structure has a characteristic chemokine fold and is in excellent agreement with the individual subunits observed in the crystal structures of dimeric SDF-1alpha. Using various peptides derived from the N-terminal extracellular tail of the CXCR4 receptor, we show that the principal determinants of binding reside in the N-terminal 17 residues of CXCR4, with a major contribution from the first six residues. From 15N/1HN chemical shift pertubation studies we show that the interaction surface on SDF-1alpha is formed by the undersurface of the three-stranded antiparallel beta-sheet bounded by the N-terminal loop on one side and the C-terminal helix on the other. This surface overlaps with but is not identical to that mapped on several other chemokines for the binding of equivalent peptides derived from their respective receptors.

Cofactor‐Apoprotein Hydrogen Bonding in Oxidized and Fully Reduced Flavodoxin Monitored by Trans‐Hydrogen‐Bond Scalar Couplings

Hydrogen bonding plays a key role in the tight binding of the FMN cofactor and the regulation of its redox properties in flavodoxins. Hydrogen bonding interactions can be directly observed in solution by multidimensional heteronuclear NMR spectroscopy through the scalar couplings between donor and acceptor nuclei. Here we report on the detection of intermolecular trans-hydrogen-bond couplings ((h)J) between the flavin ring system and the backbone of Desulfovibrio vulgaris flavodoxin in the oxidized and the two-electron reduced states. For this purpose, experiments are adapted from pulse sequences previously applied to determining (h)J coupling constants in nucleic acid-base pairs and proteins. The resulting (h2)J(N,N), (h4)J(N,N), (h3)J(C,N), and (h1)J(H,N) couplings involve the (15)N(1), (13)C(2), and (15)N(3) nuclei of the pyrimidine moiety of FMN, whereas no such interactions are detectable for (13)C(4) and (15)N(5). Several long-range (15)N-(15)N, (13)C-(15)N, and (1)H-(15)N J-coupling constants within the flavin are obtained as "by-products". The magnitudes of both (h)J and regular J couplings are found to be dependent on the redox state. In general, good correlations between (h)J coupling constants and donor-group (1)H chemical shifts and also crystallographic donor-acceptor distances are observed.

NMR Characterization of Complex p‐Oligophenyl Scaffolds by Means of Aliasing Techniques to Obtain Resolution‐Enhanced Two‐Dimensional Spectra

AbstractThe usefulness of computer‐assisted aliasing to secure maximal resolution of signal clusters in 1H‐ and 13C‐NMR spectra (which is essential for structure determination by HMBC 2D NMR spectroscopy) in minimal acquisition time is exemplified by the complete characterization of the two complementary p‐octiphenyls 1 and 2 with complex substitution patterns. The need for digital resolution near 1 Hz/pt to dissect the extensive signal clusters in the NMR spectra of these refined oligomers excluded structure determination under routine conditions. High resolution was secured by exploiting the low signal density in the 13C dimension of HMBC spectra by using computer‐assisted aliasing to maximize signal density. Based on the observed shifts in DEPT and 1H‐decoupled 13C‐NMR spectra of 1 and 2, computer‐assisted aliasing allowed to reduce the number of required time increments by a factor of 20 to 30 compared to full‐width spectra with identical resolution. Without signal‐to‐noise constraints, this computer‐assisted aliasing reduced the acquisition time for high‐resolution NMR spectra needed for complete characterization of refined oligomers 1 and 2 by the same factor (e.g., from over a day to about an hour). With resolved signal clusters in fully aliased HSQC and HMBC spectra, unproblematic structure determination of 1 and 2 is demonstrated by unambiguous assignment of all C‐ and H‐atoms. These findings demonstrate that computer‐assisted aliasing of the underexploited 13C dimension makes extensive molecular complexity accessible by conventional multidimensional heteronuclear NMR experiments without extraordinary efforts.

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor.

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.

Structure of Human MTH1, a Nudix Family Hydrolase That Selectively Degrades Oxidized Purine Nucleoside Triphosphates

Oxygen radicals generated through normal cellular respiration processes can cause mutations in genomic and mitochondrial DNA. Human MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP and 2-hydroxy-dATP, to monophosphates, thereby preventing the misincorporation of these oxidized nucleotides during replication. Here we present the solution structure of MTH1 solved by multidimensional heteronuclear NMR spectroscopy. The protein adopts a fold similar to that of Escherichia coli MutT, despite the low sequence similarity between these proteins outside the conserved Nudix motif. The substrate-binding pocket of MTH1, deduced from chemical shift perturbation experiments, is located at essentially the same position as in MutT; however, a pocket-forming helix is largely displaced in MTH1 (approximately 9 A) such that the shape of the pocket differs between the two proteins. Detailed analysis of the pocket-forming residues enabled us to identify Asn33 as one of the key residues in MTH1 for discriminating the oxidized form of purine, and mutation of this residue modifies the substrate specificity. We also show that MTH1 catalyzes hydrolysis of 8-oxo-dGTP through nucleophilic substitution of water at the beta-phosphate.

NMR structural studies of domain 1 of receptor-associated protein.

The 39 kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds tightly to the low-density lipoprotein receptor-related protein (LRP) as well as to other members of the low-density lipoprotein receptor superfamily. The association of RAP with LRP prevents this receptor from interacting with ligands. RAP is a three-domain protein that contains two independent LRP binding sites; one located within domains 1 and 2, and one located within domain 3. As the first step toward defining the structure of the full-length protein and understanding the interaction between RAP and this family of receptors, we have determined the 3D structure of domain 1 using constraints derived from heteronuclear multi-dimensional NMR spectra, including NOEs, dihedral angles, J-couplings and chemical shifts, as well as two sets of non-correlated residual dipolar couplings measured from the protein solutions in anisotropic media of Pf1 and 6% polyacrylamide gel. The backbone C(alpha) rmsd between the current structure and a homo-nuclear NOE-based structure is about 2 A. The large rmsd mainly reflects the significant differences in helical orientation and in the structural details of the long helix (helix 2) between the two structures.

NMR Solution Structure of Mob1, a Mitotic Exit Network Protein and its Interaction with an NDR Kinase Peptide

Proteins of the Mob1/phocein family are found in all eukaryotic cells. In yeast, they are activating subunits of Dbf2-related protein kinases involved in cell cycle control. Despite the wide occurrence of these proteins, their biological functions remain poorly understood. Here we report the solution structure of the Mob1 protein from Xenopus laevis solved by heteronuclear multidimensional NMR. The structure reveals a fold constituted by a central left-handed four-helix bundle, one connecting helix, two flanking helices and a long flexible loop. The clustering of two Cys and two His residues, and zinc measurement by atomic absorption spectroscopy support the existence of a zinc ion binding site. Our NMR structure is in good agreement with the recently described X-ray structure of human Mob1-A. Chemical shift perturbations observed upon addition of a peptide encompassing the basic region of the N-terminal regulatory domain of NDR kinase were used to identify and map a specific interaction between Mob1 and this kinase. The chemical shift changes indicate that the main interaction occurs on the acidic and conserved surface of Mob1. This surface was previously hypothesized to be the interaction surface according to the X-ray structure and was identified as functionally important in yeast. Our data suggest that the NDR kinase is a functional Dbf2 homologue in animal cells and contributes to the understanding of the molecular function of Mob1 proteins.

Solution structure of the 30 kDa polysulfide-sulfur transferase homodimer from Wolinella succinogenes.

The periplasmic polysulfide-sulfur transferase (Sud) protein encoded by Wolinella succinogenes is involved in oxidative phosphorylation with polysulfide-sulfur as a terminal electron acceptor. The polysulfide-sulfur is covalently bound to the catalytic Cys residue of the Sud protein and transferred to the active site of the membranous polysulfide reductase. The solution structure of the homodimeric Sud protein has been determined using heteronuclear multidimensional NMR techniques. The structure is based on NOE-derived distance restraints, backbone hydrogen bonds, and torsion angle restraints as well as residual dipolar coupling restraints for a refinement of the relative orientation of the monomer units. The monomer structure consists of a five-stranded parallel beta-sheet enclosing a hydrophobic core, a two-stranded antiparallel beta-sheet, and six alpha-helices. The dimer fold is stabilized by hydrophobic residues and ion pairs found in the contact area between the two monomers. Similar to rhodanese enzymes, Sud catalyzes the transfer of the polysulfide-sulfur to the artificial acceptor cyanide. Despite their similar functions and active sites, the amino acid sequences and structures of these proteins are quite different.

Solution structure of the matrix attachment region-binding domain of chicken MeCP2.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional protein involved in chromatin organization and silencing of methylated DNA. MAR-BD, a 125-amino-acid residue domain of chicken MeCP2 (cMeCP2, originally named ARBP), is the minimal protein fragment required to recognize MAR elements and mouse satellite DNA. Here we report the solution structure of MAR-BD as determined by multidimensional heteronuclear NMR spectroscopy. The global fold of this domain is very similar to that of rat MeCP2 MBD and MBD1 MBD (the methyl-CpG-binding domains of rat MeCP2 and methyl-CpG-binding domain protein 1, respectively), exhibiting a three-stranded antiparallel beta-sheet and an alpha-helix alpha1. We show that the C-terminal portion of MAR-BD also contains an amphipathic helical coil, alpha2/alpha3. The hydrophilic residues of this coil form a surface opposite the DNA interface, available for interactions with other domains of MeCP2 or other proteins. Spectroscopic studies of the complex formed by MAR-BD and a 15-bp fragment of a high-affinity binding site from mouse satellite DNA indicates that the coil is also involved in protein.DNA interactions. These studies provide a basis for discussion of the consequences of six missense mutations within the helical coil found in Rett syndrome cases.

Native-like Partially Folded Conformations and Folding Process Revealed in the N-terminal Large Fragments of Staphylococcal Nuclease: A Study by NMR Spectroscopy

The N-terminal large fragments of staphylococcal nuclease (SNase), SNase110 (1–110 residues), SNase121 (1–121 residues), and SNase135 (1–135 residues), and the fragment mutants G88W110, G88W121, V66W110 and V66W121 were studied by heteronuclear multidimensional NMR spectroscopy. Ensembles of co-existent native-like partially folded and unfolded states were observed for fragments. The persistent native-like tertiary interaction drives fragments to be in partially folded states, which reveal native-like β-barrel conformations. G88W and V66W mutations modulate the extent of inherent native-like tertiary interaction in fragment molecules, and in consequence, fragment mutants fold into native-like β-subdomain conformations. In cooperation with the inherent tertiary interaction, 2 M TMAO (trimethylamine N-oxide) can promote the folding reaction of fragments through the changes of unfolding free energy, and a native-like β-subdomain conformation is observed when the chain length contains 135 residues. Heterogeneous partially folded conformations of 1–121 and 1–135 fragments due to cis and trans X-prolyl bond of Lys116–Pro117 make a non-unique folding pathway of fragments. The folding reaction of fragments can be characterized as a hierarchical process.

Lipid-bound Structure of an Apolipoprotein E-derived Peptide

Apolipoprotein (apo) E regulates plasma lipid homeostasis through its ability to interact with the low density lipoprotein (LDL) receptor family. Whereas apoE is not a ligand for receptor binding in buffer alone, interaction with lipid confers receptor recognition properties. To investigate the nature of proposed lipid binding-induced conformational changes in apoE, we employed multidimensional heteronuclear NMR spectroscopy to determine the structure of an LDL receptor-active, 58-residue peptide comprising residues 126-183 of apoE in association with the micelle-forming lipid dodecylphosphocholine (DPC). In the presence of 34 mm DPC the peptide forms a continuous amphipathic helix from Glu131 to Arg178. NMR relaxation studies of DPC-bound apoE-(126-183), in contrast to apoE-(126-183) in the presence of TFE, are consistent with an isotropically tumbling peptide in solution giving a global correlation time of approximately 12.5 ns. These data indicate that the helical peptide is curved and constrained by a lipid micelle consisting of approximately 48 DPC molecules. Although the peptide behaves as if it were tumbling isotropically, spectral density analysis reveals that residues 150-183 have more motional freedom than residues 134-149. These molecular and dynamic features are discussed further to provide insight into the structural basis for the interaction between apoE and the ligand binding repeats of the LDL receptor.