Biomedical subjects

Rolf Boelens

Publications and source records attributed to Rolf Boelens.

At least 19 recordsLinked to original sources

PubMed2007

Interactions between the toxin Kid of the bacterial parD system and the antitoxins Kis and MazE.

The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here, we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid(2)-Kis(2) tetramer up to Kis(2)-Kid(2)-Kis(2)-Kid(2)-Kis(2) decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid(2)-Kis(2)-Kid(2) heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the (1)H (15)N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF(2)-MazE(2)-MazF(2) complex. Furthermore, we demonstrate that Kid(2)-MazE(2) tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed.

Antitoxins↗

PubMed2006

Light-induced flipping of a conserved glutamine sidechain and its orientation in the AppA BLUF domain.

The AppA BLUF domain is a blue light photoreceptor containing flavin. Conserved glutamine 63 is necessary for the photocycle of the protein, and its side chain has been proposed to flip in response to blue light illumination. Recently published crystal structures of AppA WT and the AppA mutant C20S describe contradictory conclusions regarding the orientation of the conserved glutamine 63 side chain in the dark. Here, we present evidence from NMR spectroscopy confirming light-induced flipping of the glutamine side chain to form a strong hydrogen bond between the glutamine 63 side chain carbonyl group and the tyrosine 21 side chain hydroxyl proton in the light-induced state. Our conclusions are consistent with published data from UV/vis absorbance and FTIR spectroscopy, as well as the crystal structure of AppA WT.

Bacterial Proteins↗

PubMed2006

The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor.

Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3',5'-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.

Binding Sites↗

PubMed2006

Solution structure of a chemosensory protein from the desert locust Schistocerca gregaria.

Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors. In this paper, we report on the solution structure of CSPsg4, a chemosensory protein from the desert locust Schistocerca gregaria, which is expressed in the antennae and other chemosensory organs. The 3D NMR structure revealed an overall fold consisting of six alpha-helices, spanning residues 13-18, 20-31, 40-54, 62-78, 80-90, and 97-103, connected by loops which in some cases show dihedral angles typical of beta-turns. As in the only other chemosensory protein whose structure has been solved so far, namely, CSP from the moth Mamestra brassicae, four helices are arranged to form a V-shaped motif; another helix runs across the two V's, and the last one is packed against the external face. Analysis of the tertiary structure evidenced multiple hydrophobic cavities which could be involved in ligand binding. In fact, incubation of the protein with a natural ligand, namely, oleamide, produced substantial changes to the NMR spectra, suggesting extensive conformational transitions upon ligand binding.

Amino Acid Sequence↗

PubMed2006

Comparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase.

In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and stability for use in washing powders. Our NMR analyses indicate virtually identical structures but reveal remarkable differences in local dynamics, with striking correspondence between experimental data (i.e., (15)N relaxation and H/D exchange rates) and data from Molecular Dynamics simulations. While overall the cores of both proteins are very rigid on the pico- to nanosecond timescale and are largely protected from H/D exchange, the two point mutations stabilize helices alpha1, alpha4, and alpha5 and locally destabilize the H-bond network of the beta-sheet (beta7-beta9). In particular, it emerges that helix alpha5, undergoing some fast destabilizing motions (on the pico- to nanosecond timescale) in wild-type lipase, is substantially rigidified by the mutation of Phe180 for a proline at its N terminus. This observation could be explained by the release of some penalizing strain, as proline does not require any "N-capping" hydrogen bond acceptor in the i+3 position. The combined experimental and simulated data thus indicate that reduced molecular flexibility of the F180P/S205G mutant lipase underlies its increased stability, and thus reveals a correlation between microscopic dynamics and macroscopic thermodynamic properties. This could contribute to the observed altered enzyme activity, as may be inferred from recent studies linking enzyme kinetics to their local molecular dynamics.

Amino Acid Sequence↗

PubMed2006

Information-driven protein-DNA docking using HADDOCK: it is a matter of flexibility.

Intrinsic flexibility of DNA has hampered the development of efficient protein-DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731-1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor-DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein-DNA complexes from unbound constituents using non-structural experimental data to drive the docking.

Computational Biology↗

PubMed2006

Direct use of unassigned resonances in NMR structure calculations with proxy residues.

We present a method that significantly enhances the robustness of (automated) NMR structure determination by allowing the NOE data corresponding to unassigned NMR resonances to be used directly in the calculations. The unassigned resonances are represented by additional atoms or groups of atoms that have no interaction with the regular protein atoms except through distance restraints. These so-called "proxy" residues can be used to generate NOE-based distance restraints in a similar fashion as for the assigned part of the protein. If sufficient NOE information is available, the restraints are expected to place the proxies at positions close to the correct atoms for the unassigned resonance, which can facilitate subsequent assignment. Convergence can be further improved by supplying additional information about the possible identities of the unassigned resonances. We have implemented this approach in the widely used automated assignment and structure calculation protocols ARIA and CANDID. We find that it significantly increases the robustness of structure calculations with regard to missing assignments and yields structures of higher quality. Our approach is still able to find correctly folded structures with up to 30% randomly missing resonance assignments, and even when only backbone and beta resonances are present! This should be of significant value to NMR-based structural proteomics initiatives.

Journal Article↗

PubMed2006

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange.

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.

Bacterial Proteins↗

PubMed2006

The intrinsically unstructured domain of PC4 modulates the activity of the structured core through inter- and intramolecular interactions.

Proteins frequently contain unstructured regions apart from a functionally important and well-conserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, nuclear magnetic resonance (NMR), and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd.

Binding Sites↗

PubMed2006

The solution structure of the AppA BLUF domain: insight into the mechanism of light-induced signaling.

The transcriptional antirepressor AppA from the photosynthetic bacterium Rhodobacter sphaeroides senses both the light climate and the intracellular redox state. Under aerobic conditions in the dark, AppA binds to and thereby blocks the function of PpsR, a transcriptional repressor. Absorption of a blue photon dissociates AppA from PpsR and allows the latter to repress photosynthesis gene expression. The N terminus of AppA contains sequence homology to flavin-containing photoreceptors that belong to the BLUF family. Structural and chemical aspects of signal transduction mediated by AppA are still largely unknown. Here we present NMR studies of the N-terminal flavin-binding BLUF domain of AppA. Its solution structure adopts an alpha/beta-sandwich fold with a beta alpha beta beta alpha beta beta topology, which represents a new flavin-binding fold. Considerable disorder is observed for residues near the chromophore due to conformational exchange. This disorder is observed both in the dark and in the light-induced signaling state of AppA. Furthermore, we detect light-induced structural changes in a patch of surface residues that provide a structural link between light absorption and signal-transduction events.

Bacterial Proteins↗

PubMed2006

Combining NMR relaxation with chemical shift perturbation data to drive protein-protein docking.

The modeling of biomolecular complexes by computational docking using the known structures of their constituents is developing rapidly to become a powerful tool in structural biology. It is especially useful in combination with even limited experimental information describing the interface. Here we demonstrate for the first time the use of diffusion anisotropy in combination with chemical shift perturbation data to drive protein-protein docking. For validation purposes we make use of simulated diffusion anisotropy data. Inclusion of this information, which can be derived from NMR relaxation rates and reports on the orientation of the components of a complex with respect to the rotational diffusion tensor, substantially improves the docking results.

Anisotropy↗

PubMed2005

Model for RNA binding and the catalytic site of the RNase Kid of the bacterial parD toxin-antitoxin system.

The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

Bacterial Proteins↗

PubMed2005

Solution structure of the C1-subdomain of Bacillus stearothermophilus translation initiation factor IF2.

IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet-tRNAf(Met). After these interactions, fMet-tRNAf(Met) is oriented to the ribosomal P-site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C-terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet-tRNAf(Met), contains two structured modules. Previously, the solution structure of the most C-terminal module, IF2-C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet-tRNAf(Met) were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C-terminal domain (IF2-C1) and determined its solution structure and backbone dynamics. The IF2-C1 core forms a flattened fold consisting of a central four-stranded parallel beta-sheet flanked by three alpha-helices. Although its overall organization resembles that of subdomain III of the archaeal IF2-homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident.

Eukaryotic Initiation Factor-2↗

PubMed2005

Dynamics and metal exchange properties of C4C4 RING domains from CNOT4 and the p44 subunit of TFIIH.

Zinc fingers are small structured protein domains that require the coordination of zinc for a stable tertiary fold. Together with FYVE and PHD, the RING domain forms a distinct class of zinc-binding domains, where two zinc ions are ligated in a cross-braced manner, with the first and third pairs of ligands coordinating one zinc ion, while the second and fourth pairs ligate the other zinc ion. To investigate the relationship between the stability and dynamic behaviour of the domains and the stability of the metal-binding site, we studied metal exchange for the C4C4 RING domains of CNOT4 and the p44 subunit of TFIIH. We found that Zn(2+)-Cd(2+) exchange is different between the two metal-binding sites in the C4C4 RING domains of the two proteins. In order to understand the origins of these distinct exchange rates, we studied the backbone dynamics of both domains in the presence of zinc and of cadmium by NMR spectroscopy. The differential stability of the two metal-binding sites in the RING domains, as reflected by the different metal exchange rates, can be explained by a combination of accessibility and an electrostatic ion interaction model. A greater backbone flexibility for the p44 RING domain as compared to CNOT4 may be related to the distinct types of protein-protein interactions in which the two C4C4 RING domains are involved.

Amino Acid Sequence↗

PubMed2005

Solution structure of the C-terminal domain of TFIIH P44 subunit reveals a novel type of C4C4 ring domain involved in protein-protein interactions.

The human general transcription factor TFIIH is involved in both transcription and DNA nucleotide excision repair. Among the 10 subunits of the complex, p44 subunit plays a crucial role in both mechanisms. Its N-terminal domain interacts with the XPD helicase, whereas its C-terminal domain is involved specifically in the promoter escape activity. By mutating an exposed and non-conserved cysteine residue into a serine, we produced a soluble mutant of p44-(321-395) suitable for solution structure determination. The domain adopts a C4C4 RING domain structure with sequential organization of beta-strands that is related to canonical RING domains by a circular permutation of the beta-sheet elements. Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321-395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, thus, differs from previously reported interaction mechanisms involving RING domains.

Amino Acid Sequence↗

PubMed2005

Structural properties of the promiscuous VP16 activation domain.

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

Herpes Simplex Virus Protein Vmw65↗

PubMed2005

Altered specificity in DNA binding by the lac repressor: a mutant lac headpiece that mimics the gal repressor.

Recognition of the lac operator by the lac repressor involves specific interactions between residues in the repressor's recognition helix and bases in the DNA major groove. Tyr17 and Gln18, at positions 1 and 2 in the lac repressor recognition helix, can be exchanged for other amino acids to generate mutant repressors that display altered specificity. We have solved the solution structure of a protein-DNA complex of an altered-specificity mutant lac headpiece in which Tyr17 and Gln18 were exchanged for valine and alanine, respectively, as found in the recognition helix of the gal repressor. As previously described by Lehming et al. (EMBO J. 1987, 6, 3145-3153), this altered-specificity mutant of the lac repressor recognizes a variant lac operator that is similar to the gal operator Oe. The mutant lac headpiece showed the predicted specificity and is also able to mimic the gal repressor by recognizing and bending the natural gal operator Oe. These structural data show that, while most of the anchoring points that help the lac headpiece to assemble on the lac operator were preserved, a different network of protein-DNA interactions connecting Ala17 and Val18 to bases in the DNA major groove drives the specificity towards the altered operator.

Amino Acid Sequence↗

PubMed2005

Describing partially unfolded states of proteins from sparse NMR data.

Proteins involved in signal transduction can usually be present in two states: an inactive and an active (signaling) state. In the case of photoreceptors such as PYP, it has been shown that the signaling state has a large degree of structural and dynamic disorder. Conventional structural NMR approaches present difficulties in describing such partially unfolded states. Owing to the disordered dynamical and transient nature of such states classical NOE-based information, when present, is sparse. Chemical shift changes upon partial unfolding can, however, be easily monitored from HSQC spectra. We show here that such states can be modeled by defining native-like inter-residue contacts for those residues that do not shift significantly upon partial unfolding. The feasibility of this approach is demonstrated using lysozyme as a test case and applied to model the partially unfolded signaling state (pB) of a truncated form of the photoactive yellow protein for which a "classical" NOE-based structure is available for validation. This approach should be generally applicable to systems in which part of the structure remains in a well-defined native-like conformation.

Bacterial Proteins↗