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Biomedical subjects

R Boelens

Publications and source records attributed to R Boelens.

At least 37 records · Page 2Linked to original sources
PubMed1999

Millisecond to microsecond time scale dynamics of the retinoid X and retinoic acid receptor DNA-binding domains and dimeric complex formation.

The all-trans retinoic acid and 9-cis retinoic acid receptors (RAR and RXR, respectively) belong to a family of ligand inducible transcription factors, which exert their effect via binding to hormone response elements. Both are members of the class II sub-family of nuclear receptors, which bind DNA as dimers, on tandem repeats of a hexamer motif separated by a variable spacer. The variability in spacer length and the head-to-tail organization of the hormone response elements result in different protein-protein interactions in each of the complexes. We show that the zinc-coordinating loop regions of RXR and RAR DNA-binding domains exhibit dynamics on the millisecond to microsecond time scale. The highly dynamic second zinc finger of RXR constitutes the primary protein-protein interface in many nuclear receptor assemblies on DNA. Dynamics is also observed in the first and second zinc fingers of RAR, which are implicated in dimeric interactions with RXR on response elements with spacers of 5 base pairs and 1 base pair, respectively. The striking correspondence between the regions that exhibit conformational exchange and the dimer interfaces of the proteins complexed with DNA suggests a functional role for the dynamics. The observed flexibility may allow the proteins to adapt to various partners and with different orientations upon assembly on DNA. Furthermore, the more extensive dynamics observed for RXR may reflect the greater ability of this protein to modulate its interaction surface since it participates in a wide variety of receptor complexes.

Amino Acid Sequence↗
PubMed1999

Identification of the single-stranded DNA binding surface of the transcriptional coactivator PC4 by NMR.

The C-terminal domain of the eukaryotic transcriptional cofactor PC4 (PC4CTD) is known to bind with nanomolar affinity to single-stranded (ss)DNA. Here, NMR is used to study DNA binding by this domain in more detail. Amide resonance shifts that were observed in a 1H15N-HSQC-monitored titration of 15N-labeled protein with the oligonucleotide dT18 indicate that binding of the nucleic acid occurs by means of two anti-parallel channels that were previously identified in the PC4CTD crystal structure. The beta-sheets and loops that make up these channels exhibit above average flexibility in the absence of ssDNA, which is reflected in higher values of T1rho, reduced heteronuclear nuclear Overhauser effects and faster deuterium exchange rates for the amides in this region. Upon ssDNA binding, this excess flexibility is significantly reduced. The binding of ssDNA by symmetry-related channels reported here provides a structural rationale for the preference of PC4CTD for juxtaposed single-stranded regions (e.g. in heteroduplexes) observed in earlier work.

Binding Sites↗
PubMed1999

Editing of chemical exchange-relayed NOEs in NMR experiments for the observation of protein-water interactions.

An experimental approach for the editing of exchange-relayed NOEs in water-selective NOE experiments is presented. The proposed pulse sequence is based on the application during the NOE mixing time of continuous wave irradiation, which saturates resonances of relaying labile protons in slow chemical exchange with water. The technique can efficiently reduce the contributions of exchange-relayed NOE peaks that often crowd the water-selective NOE spectra and hide direct intermolecular NOEs between water and protein protons. The present approach opens new opportunities for the characterization of hydration by NMR, even in the proximity of polar labile groups.

Macromolecular Substances↗
PubMed1999

The role of high-resolution structural studies in the development of commercial enzymes.

Recent developments in both NMR and X-ray crystallography allow the analysis of commercial enzymes in unprecedented detail. The novel methods provide detailed insights into protein dynamics, establish the existence of special catalytic hydrogen bonds and define the ionization states at the enzyme active site. A more detailed understanding of how the changes in structure are related to altered function should facilitate the design of future commercial enzymes with improved performance for different environmental conditions.

Biotechnology↗
PubMed1999

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy.

Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional (15N,1H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected. Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and alpha-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain. The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor.

Amino Acid Sequence↗
PubMed1999

Water-macromolecule interactions by NMR: a quadrature-free constant-time approach and its application to CI2.

A pulse sequence is proposed to select water magnetization with enhanced specificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented without quadrature detection, which results in a square root 2 increase in signal-to-noise ratios as compared to traditional non-selective methods for water filtration. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gains in sensitivity. The proposed experiment was applied to unlabeled HEW lysozyme and to 15N-labeled chymotrypsin inhibitor 2 which was partially or non 13C-enriched. Water molecules belonging to a spine of hydration between two pseudo beta-sheet strands were identified, solving previously reported discrepancies between the X-ray and refined NMR structure of CI2. The proposed experiment in particularly suitable for hydration studies of mixtures of labeled and unlabeled components, such as ligand-macromolecule complexes.

Hydrogen Bonding↗
PubMed1999

Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements.

Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1 rho experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351-357] were applied to the 9-cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 microseconds, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271-284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.

Amino Acid Sequence↗
PubMed1999

The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils.

The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.

Amino Acid Sequence↗
PubMed1999

Solution structure of the alpha-subunit of human chorionic gonadotropin.

The three-dimensional solution structure of the alpha-subunit in the alpha, beta heterodimeric human chorionic gonadotropin (hCG), deglycosylated with endo-beta-N-acetylglucosaminidase-B (dg-alpha hCG), was determined using 2D homonuclear and 2D heteronuclear 1H, 13C NMR spectroscopy at natural abundance in conjunction with the program package XPLOR. The distance geometry/simulated annealing protocol was modified to allow for the efficient modelling of the cystine knot motif present in alpha hCG. The protein structure was modelled with 620 interproton distance restraints and the GlcNAc residue linked to Asn78 was modelled with 30 protein-carbohydrate and 3 intraresidual NOEs. The solution structure of dg-alpha hCG is represented by an ensemble of 27 structures. In comparison to the crystal structure of the dimer, the solution structure of free dg-alpha hCG exhibits: (a) an increased structural disorder (residues 33-57); (b) a different backbone conformation near Val76 and Glu77; and (c) a larger flexibility. These differences are caused by the absence of the interactions with the beta-subunit. Consequently, in free dg-alpha hCG, compared to the intact dimer, the two hairpin loops 20-23 and 70-74 are arranged differently with respect to each other. The beta-GlcNAc(78) is tightly associated with the hydrophobic protein-core in between the beta-hairpins. This conclusion is based on the NOEs from the axial H1, H3, H5 atoms and the N-acetyl protons of beta-GlcNAc(78) to the protein-core. The hydrophobic protein-core between the beta-hairpins is thereby shielded from the solvent.

Crystallography, X-Ray↗
PubMed1998

Solution structure and backbone dynamics of the photoactive yellow protein.

The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.

Bacterial Proteins↗
PubMed1998

Mobilities of the inner three core residues and the Man(alpha 1--6) branch of the glycan at Asn78 of the alpha-subunit of human chorionic gonadotropin are restricted by the protein.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free alpha-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991) Endocrinology 129, 2257-2259]. Glycosylation at Asn78 of the alpha-subunit is required for the stability of the protein, but the exact nature of the stabilizing effect is not known. In our previous study, it was indicated that GlcNAc-1 at Asn78 has a reduced mobility, whereas the glycan at Asn52 is highly mobile [De Beer, T., et al. (1996) Eur. J. Biochem. 241, 229-242]. In the present investigation, it is shown that the PNGase F susceptibility of the Asn52-linked glycan in the free alpha-subunit is absent in the heterodimer. Thus, the high mobility of the glycan at Asn52 may be characteristic for the free alpha-subunit. For accurate modeling of alpha hCG, knowledge of the behavior of each of the glycans is essential. In this context, the mobility of the glycans and their interactions with the protein are explored by NMR spectroscopy using desialylated, partially deglycosylated free alpha-subunit (as-pd alpha) carrying glycans at Asn78 only. NOEs between GlcNAc-2 and several amino acid residues indicate that GlcNAc-2 is involved in stabilizing alpha hCG. From the values of 13C relaxation parameters T2 and T1 rho of the constituting monosaccharide residues, it was concluded that the inner three residues have a severely restricted mobility. The Man-4 and Man-4' residues of the diantennary oligosaccharide exhibit a similar relaxation behavior, suggesting that the Man-4' branch occurs in a single conformation of the C5-C6 linkage of Man-3 instead of in rapidly interconverting conformations that are known to exist for this linkage for the free oligosaccharide.

Asparagine↗
PubMed1998

An Off-resonance Rotating Frame Relaxation Experiment for the Investigation of Macromolecular Dynamics Using Adiabatic Rotations

15N off-resonance rotating frame relaxation can be applied to the study of internal dynamics in proteins in the millisecond to microsecond regime. We show that the performance of existing methods can be improved by application of simultaneous amplitude and phase-modulated adiabatic RF pulses to align the nuclear spin magnetization with the off-resonance spin-lock field for all the spins under investigation. Application of this technique to the 269-residue serine protease PB92 allowed the measurement of 15N off-resonance rotating frame relaxation rates for all nonoverlapping residues in the protein, including the arginine side chains, encompassing a chemical shift range of 50 ppm. Simulations indicate that by use of the proposed adiabatic RF pulses rotating frame relaxation rates can be obtained for magnetization vectors aligned at arbitrary angles with the static field. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Sensitivity enhancement in the TROSY experiment.

A modification of the TROSY experiment which allows for sensitivity enhancement without introduction of additional delays is presented. The method relies on the combination of two orthogonal pathways and results in a signal-to-noise gain of 2 independent of the size of the molecule. The sensitivity enhanced TROSY scheme can be combined with gradient coherence selection. The method has been applied to the 269 residue serine protease PB92.

Glycine↗
PubMed1998

Structural and dynamic changes of photoactive yellow protein during its photocycle in solution.

Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.

Bacterial Proteins↗
PubMed1997

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc.

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Amino Acid Sequence↗
PubMed1997

The solution structure of serine protease PB92 from Bacillus alcalophilus presents a rigid fold with a flexible substrate-binding site.

BACKGROUND: Research on high-alkaline proteases, such as serine protease PB92, has been largely inspired by their industrial application as protein-degrading components of washing powders. Serine protease PB92 is a member of the subtilase family of enzymes, which has been extensively studied. These studies have included exhaustive protein engineering investigations and X-ray crystallography, in order to provide insight into the mechanism and specificity of enzyme catalysis. Distortions have been observed in the substrate-binding region of subtilisin crystal structures, due to crystal contacts. In addition, the structural variability in the substrate-binding region of subtilisins is often attributed to flexibility. It was hoped that the solution structure of this enzyme would provide further details about the conformation of this key region and give new insights into the functional properties of these enzymes. RESULTS: The three-dimensional solution structure of the 269-residue (27 kDa) serine protease PB92 has been determined using distance and dihedral angle constraints derived from triple-resonance NMR data. The solution structure is represented by a family of 18 conformers which overlay onto the average structure with backbone and all-heavy-atom root mean square deviations (for the main body of the molecule) of 0.88 and 1.21 A, respectively. The family of structures contains a number of regions of relatively high conformational heterogeneity, including various segments that are involved in the formation of the substrate-binding site. The presence of flexibility within these segments has been established from NMR relaxation parameters and measurements of amide proton exchange rates. CONCLUSIONS: The solution structure of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites. Among the limited number of residues involved in significant internal mobility are those of two pockets, termed S1 and S4, within the substrate-binding site. The presence of flexibility within the binding site supports the proposed induced fit mechanism of substrate binding.

Amino Acid Sequence↗
PubMed1997

The structure of the translational initiation factor IF1 from E.coli contains an oligomer-binding motif.

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root-mean-square deviation versus the average of 0.49 A for the backbone atoms and 1.12 A for all atoms for residues 6-36 and 46-67. The structure of IF1 is characterized by a five-stranded beta-barrel. The loop connecting strands three and four contains a short 3(10) helix but this region shows considerably higher flexibility than the beta-barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N-terminal domain of aspartyl-tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer-binding motif. Several proteins of this family are nucleic acid-binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes.

Amino Acid Sequence↗