Detection of the protein-protein interaction between cyclic AMP receptor protein and RNA polymerase, by (13)C-carbonyl NMR.

Cyclic AMP receptor protein (CRP) plays a key role in the transcription regulation of many prokaryotic genes. Upon the binding of cyclic AMP, CRP is allosterically activated, binds to target DNA sites, and interacts with RNA polymerase. Although the protein-protein interaction between CRP and RNA polymerase is known to be important for the transcription initiation of the target genes, its structural understanding is still lacking, particularly due to the high molecular mass (approximately 120 kDa) of the protein complex. We assigned all of the (13)C-carbonyl resonances of methionine residues in CRP by using the double labeling and the enzyme digestion techniques. The result of (13)C-carbonyl NMR experiment on [(13)C'-Met]-CRP in the presence of both cyclic AMP and RNA polymerase alpha subunit showed that the two proteins interact with each other in solution in the absence of DNA via the region around the residues from Met 157 to Met 163 in CRP. The results also showed the effectiveness of the selective labeling and (13)C-carbonyl NMR spectroscopy in the specific detection of the protein-protein interaction between large molecules.

Conformational study on poly [gamma-(alpha-phenethyl)-L-glutamate] using vibrational circular dichroism spectroscopy.

Vibrational circular dichroism (VCD) and IR absorption spectra are obtained in a chloroform solution for poly[gamma-((R)-alpha-phenethyl)-L-glutamate] (PRPLG) and poly[gamma-((S)-alpha-phenethyl)-L-glutamate] (PSPLG), whose only structural difference is an opposite chiral center in the side chain. Their characteristic amide A, I, and II bands show VCD patterns quite similar to those of poly[gamma-benzyl-L-glutamate] (PBLG), indicating that the secondary structure of these polypeptides is a right-handed alpha-helix. The VCD spectra in the CH stretching region exhibit different patterns for PRPLG and PSPLG, reflecting the chirality difference in the side chains. This difference is interpreted on the basis of the additivity of optical activity contributions from the main chain conformation and the chirality difference in the side chains. The results indicate that a VCD difference spectrum of the CH stretching region is a useful diagnostic tool for elucidating local chirality differences.

Interaction of the C-terminal domain of the E. coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface.

The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA. Previously, we determined the solution structure of alphaCTD. Here, we investigated the interaction between alphaCTD and UP element DNA by NMR. DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA. Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding. There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay. We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending. Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA. The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction. Based on these observations, we propose a model for the UP element-alphaCTD complex.

Sugar conformation of a stereospecific 2'-R or 2'-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants.

The sugar conformation of a DNA decamer was studied with proton-proton 3J coupling constants. Two samples, one comprising stereospecifically labeled 2'-R-2H for all residues and the other 2'-S-2H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980-6986; Nucleosides Nucleotides (1995) 14, 333-336], the deuterium labeling being highly stereospecific (> or = 99% for all 2''-2H, > or = 98% for 2'-2H of A, C, and T, and > or = 93% for 2'-2H of G). The 3J values of all H1'-H2' and H1'-H2'' pairs, and several H2'-H3' and H2''-H3' pairs were determined by line fitting of 1D spectra with 0.1-0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3'-exo and C2'-endo with phi(m) values of 26 degrees to 44 degrees, except for the second and 3' terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from JH1'H2' and JH1'H2'' values. For C10, the N-S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1'-exo conformation with 27 degrees distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.

Structural genomics projects in Japan.

Two major structural genomics projects exist in Japan. The oldest, the RIKEN Structural Genomics Initiative, has two major goals: to determine bacterial, mammalian, and plant protein structures by X-ray crystallography and NMR spectroscopy and to perform functional analyses with the target proteins. The newest, the structural genomics project at the Biological Information Research Center, focuses on human membrane proteins.

Structural understanding of the allosteric conformational change of cyclic AMP receptor protein by cyclic AMP binding.

Cyclic AMP receptor protein (CRP) plays a key role in the regulation of more than 150 genes. CRP is allosterically activated by cyclic AMP and binds to specific DNA sites. A structural understanding of this allosteric conformational change, which is essential for its function, is still lacking because the structure of apo-CRP has not been solved. Therefore, we performed various NMR experiments to obtain apo-CRP structural data. The secondary structure of apo-CRP was determined by analyses of the NOE connectivities, the amide proton exchange rates, and the (1)H-(15)N steady-state NOE values. A combination of the CSI-method and TALOS prediction was also used to supplement the determination of the secondary structure of apo-CRP. This secondary structure of apo-CRP was compared with the known structure of cyclic AMP-bound CRP. The results suggest that the allosteric conformational change of CRP caused by cyclic AMP binding involves subunit realignment and domain rearrangement, resulting in the exposure of helix F onto the surface of the protein. Additionally, the results of the one-dimensional [(13)C]carbonyl NMR experiments show that the conformational change of CRP caused by the binding of cyclic GMP, an analogue of cyclic AMP, is different from that caused by cyclic AMP binding.

Three-dimensional solution structure of a disulfide bond isomer of the human insulin-like growth factor-I.

The solution structure of a disulfide bond isomer of human insulin-like growth factor-I (IGF-I) was determined using homonuclear NMR methods. A total of 292 interatomic distance constraints, including 12 related to the disulfide bridges, was used in the distance geometry calculations. The determined structures contain two helical rods corresponding to the sequence regions, Ala8-Cys18 and Leu54-Cys61. Comparison with the previously determined structure of native human IGF-I revealed partial correspondence of the secondary structure (helices I: Ala8-Cys18 and helices III: Leu54-Cys61) and internal packing. Helix II in native human IGF-I (residues Gly42-Cys48) is disrupted in the isomer. A similar relationship has been described between the structure of native insulin and a homologous disulfide isomer, suggesting that these alternative folds represent general features of insulin-like sequences. In each case the precision of the distance geometry ensemble is low due in part to resonance broadening and a paucity of NOEs relative to other globular proteins of this size. These observations suggest that tertiary structure of the isomer is not highly ordered. Comparison of the biological activities of native and the disulfide bond isomer of human IGF-I highlight the importance of Tyr24, Phe25, Phe49-Cys52 and Phe16 in binding to the IGF-I receptor or specific IGFBPs. The relationship of this proposed receptor-binding surface of human IGF-I to those of insulin is discussed.

Structural study of the N-terminal domain of the alpha subunit of Escherichia coli RNA polymerase solubilized with non-denaturing detergents.

The amino-terminal domain of the alpha subunit (alphaNTD) of Escherichia coli RNA polymerase consisting of 235 amino acid residues functions in the assembly of the alpha, beta, and beta' subunits into the core-enzyme. It has a tendency to form aggregates by itself at higher concentrations. For NMR structural analysis of alphaNTD, the solution conditions, including the use of non-denaturing detergents, were optimized by monitoring the translational diffusion coefficients using the field gradient NMR technique. Under the optimal conditions with taurodeoxycholate and with the aid of deuteration of the sample, alphaNTD gave triple-resonance spectra of good quality, which allowed the assignment of a large part of the backbone resonances. Analysis of the pattern of NOEs observed between the backbone amide and alpha-protons demonstrated that alphaNTD has three alpha-helices and two beta-sheets. Although the secondary structure elements essentially coincide with those in the crystal structure, the larger of the two beta-sheets has two additional beta-strands. The irregular NOE patterns observed for the three positions in the beta-sheets suggest the presence of beta-bulge structures. The positions of the three helices coincide with the conserved sequence regions that are responsible for the subunit assembly.

Solution structure of an RNA duplex including a C-U base pair.

The formation of the C-U base pair in a duplex was observed in solution by means of the temperature profile of (15)N chemical shifts, and the precise geometry of the C-U base pair was also determined by NOE-based structure calculation. From the solution structure of the RNA oligomer, r[CGACUCAGG].r[CCUGCGUCG], it was found that a single C-U mismatch preferred being stacked in the duplex rather than being flipped-out even in solution. Moreover, it adopts an irregular geometry, where the amino nitrogen (N4) of the cytidine and keto-oxygen (O4) of the uridine are within hydrogen-bonding distance, as seen in crystals. To further prove the presence of a hydrogen bond in the C-U pair, we employed a point-labeled cytidine at the exocyclic amino nitrogen of the cytidine in the C-U pair. The temperature profile of its (15)N chemical shift showed a sigmoidal transition curve, indicating the presence of a hydrogen bond in the C-U pair in the duplex.

The structure and the characteristic DNA binding property of the C-terminal domain of the RNA polymerase alpha subunit from Thermus thermophilus.

The C-terminal domain of the alpha subunit of the RNA polymerase (alphaCTD) from Escherichia coli (Ec) regulates transcription by interacting with many kinds of proteins and promoter upstream (UP) elements consisting of AT-rich sequences. However, it is unclear how this system is common in all eubacteria. We investigate the structure and properties of alphaCTD from an extremely thermophilic eubacterium, Thermus thermophilus (Tt). The solution structure of Tt alphaCTD (85 amino acids) was determined by NMR, and the interaction between Tt alphaCTD and DNA with different sequences was investigated by means of chemical shift perturbation experiments. The tertiary structure of Tt alphaCTD is almost identical with that of Ec alphaCTD despite 32% sequence homology. However, Tt alphaCTD interacts with the upstream region sequence of the promoter in the Tt 16 S ribosomal protein operon rather than the Ec UP element DNA. The upstream region sequence of Tt is composed of 25 base pairs with 40% AT, unlike the Ec UP element with 80% AT. The DNA binding site in Tt alphaCTD is located on the surface composed of helix 4 and the loop preceding helix 4. The electric charges on this surface are not remarkably localized like those of Ec alphaCTD.

Structure of the CAD domain of caspase-activated DNase and interaction with the CAD domain of its inhibitor.

Caspase-activated DNase (CAD), which causes a genome fragmentation at the final stage of apoptosis, is a protein of about 40 kDa and exists as a complex form with the inhibitor ICAD in living cells. There is sequence homology of about 80 amino acid residues at the N termini of CAD and ICAD (called the CAD domain). Here, we report the three-dimensional structure of the CAD domain of CAD determined by multi-dimensional NMR spectroscopy and the property of CAD domains investigated by a surface plasmon resonance experiment. The CAD domain of CAD is an independently folded domain composed of one alpha-helix and five beta-strands forming a single sheet. The overall structure is categorized in the ubiquitin superfold. This domain can bind strongly to the isolated CAD domain of ICAD (dissociation constant: 5.48(+/-0.003)x10(-8) M). It suggests the function of the CAD domains in the CAD-ICAD system, that the protein-protein interaction through the CAD domains plays an important role in the inhibition of CAD DNase activity and in the correct folding of CAD. On the basis of structural comparison with other protein complexes containing the ubiquitin superfold, the interaction mode of the CAD domains is proposed.

NMR observation of selected segments in a larger protein: central-segment isotope labeling through intein-mediated ligation.

Peptide segments in a protein, which can include an active site of interest or be a series of parts constituting the entire structure, are now selectively observed by nuclear magnetic resonance (NMR) spectroscopy using samples prepared by the intein-mediated ligation method. Two separate inteins were used to ligate NMR-transparent segments to both the ends of an NMR-visible segment, producing a partly visible intact protein molecule. The (15)N-(1)H correlation spectrum of a 370-residue maltose binding protein labeled with (15)N at a continuous segment comprising residues Gly(101)-Ser(238) showed the essential elimination of signal overlapping, the signals being at the same positions as for the uniformly labeled sample. This method will allow structural analysis by NMR of over 50-kDa proteins in combination with contemporary NMR techniques suppressing the signal decays of larger proteins.

Crystal structure of an IRF-DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences.

There has been growing interest in the role of the IRF (interferon regulatory factor) family of transcription factors in the regulation of immune responses, cytokine signaling, and oncogenesis. These members are characterized by their well-conserved DNA binding domains at the N-terminal regions. Here we report the 2.2 A resolution crystal structure of the DNA binding domain of one such family member, IRF-2, bound to DNA. The structure reveals its recognition sequence, AANNGAAA (here, recognized bases are underlined and in bold, and N indicates any base), and its cooperative binding to a tandem repeat of the GAAA core sequence induced by DNA structure distortions. These facts explain well the diverse binding properties of the IRF family members, which bind to both single and tandemly repeated sequences. Furthermore, we also identified the 'helix-hairpin-strand motif' at the C terminus of the recognition helix as a metal binding site that is commonly found in certain classes of DNA-interactive proteins. Our results provide new insights into the structure and function of this family of transcription factors.

Improved segmental isotope labeling of proteins and application to a larger protein.

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.

Intermolecular contacts between the lambda-Cro repressor and the operator DNA characterized by nuclear magnetic resonance spectroscopy.

The specific interaction between lambda phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both 15N- and 13C/15N- labeled lambda-Cro in complex with unlabeled DNA, chemical shift assignments of the lambda-Cro-DNA complex were obtained using heteronuclear NMR experiments. Inter-molecular contacts between the protein and DNA were identified using heteronuclear filtered NOESY experiments. The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOE constraints to dock lambda-Cro to the bent B-form DNA using restrained molecular dynamics. The structure of one of the subunits of lambda-Cro in the complex is essentially the same as that of the unbound form. In the complex, inter-molecular NOEs were observed between the "helix-turn-helix" region comprising the alpha2 and alpha3 helices of the lambda-Cro protein and the major groove of the DNA. The methyl group of Thr17 forms a hydrophobic contact with the methyl group of the thymine at base pair 1 in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5. The presence and the absence of these contacts can explain the difference in the affinity of lambda-Cro to several variants of the operator sequence.

Translational induction of heat shock transcription factor sigma32: evidence for a built-in RNA thermosensor.

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock sigma-factor sigma32 encoded by the rpoH gene. Increased sigma32 levels result from both enhanced synthesis and stabilization. Previous work indicated that sigma32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5'-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of sigma32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA-30S ribosome-tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator.

Resonance assignments, solution structure, and backbone dynamics of the DNA- and RPA-binding domain of human repair factor XPA.

XPA is involved in the damage recognition step of nucleotide excision repair (NER). XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA). The domain consists of a zinc-containing subdomain with a compact globular structure and a C-terminal subdomain with a positively charged cleft in a novel alpha/beta structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods. 15N relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion. In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (Dparallel/Dperpendicular) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-terminal subdomain. This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA.

A'-form RNA double helix in the single crystal structure of r(UGAGCUUCGGCUC).

Here we demonstrate the presence of the A'-RNA conformation using the single crystal structure of a tridecamer: r(UGAGCUUCGGCUC). The average A'-RNA conformation deduced from X-ray fiber diffraction data had only been available previously, but now the presence of the A'-RNA conformation has been found in a single crystal structure for the first time. Statistical analysis showed that the A'-RNA conformation is distinguishable from the A-RNA conformation in a plot of the major groove width against the base pair inclination angle. The major groove of the A'-RNA conformation is wide enough to accommodate a protein or peptide while that of the A-RNA conformation is too narrow to do so. The presence of the A'-RNA conformation is significant for protein-RNA interaction.