Biomedical subjects

Robert Kaptein

Publications and source records attributed to Robert Kaptein.

At least 19 recordsLinked to original sources

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

Structural characterization of Spo0E-like protein-aspartic acid phosphatases that regulate sporulation in bacilli.

Spore formation is an extreme response of many bacterial species to starvation. In the case of pathogenic species of Bacillus and Clostridium, it is also a component of disease transmission. Entry into the pathway of sporulation in Bacillus subtilis and its relatives is controlled by an expanded two-component system in which starvation signals lead to the activation of sensor kinases and phosphorylation of the master sporulation response regulator Spo0A. Accumulation of threshold concentrations of Spo0A approximately P heralds the commitment to sporulation. Countering the activities of the sensor kinases are phosphatases such as Spo0E, which dephosphorylate Spo0A approximately P and inhibit sporulation. Spo0E-like protein-aspartic acid-phosphate phosphatases, consisting of 50-90 residues, are conserved in sporeforming bacteria and unrelated in sequence to proteins of known structure. Here we determined the structures of the Spo0A approximately P phosphatases BA1655 and BA5174 from Bacillus anthracis using nuclear magnetic resonance spectroscopy. Each is composed of two anti-parallel alpha-helices flanked by flexible regions at the termini. The signature SQELD motif (SRDLD in BA1655) is situated in the middle of helix alpha2 with its polar residues projecting outward. BA5174 is a monomer, whereas BA1655 is a dimer. The four-helix bundle structure in the dimer is reminiscent of the phosphotransferase Spo0B and the chemotaxis phosphatase CheZ, although in contrast to these systems, the subunits in BA1655 are in head-to-tail rather than head-to-head apposition. The implications of the structures for interactions between the phosphatases and their substrate Spo0A approximately P are discussed.

Amino Acid Motifs↗

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 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↗

PubMed2006

Gradual phosphorylation regulates PC4 coactivator function.

The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces.

Amino Acid Sequence↗

PubMed2005

Solution structure of the human ubiquitin-specific protease 15 DUSP domain.

Ubiquitin-specific proteases (USPs) can remove covalently attached ubiquitin moieties from target proteins and regulate both the stability and ubiquitin-signaling state of their substrates. All USPs contain a conserved catalytic domain surrounded by one or more subdomains, some of which contribute to target recognition. One such specific subdomain, the DUSP domain (domain present in ubiquitin-specific proteases), is present in at least seven different human USPs that regulate the stability of or interact with the hypoxia-inducible transcription factor HIF1-alpha, the Von Hippel-Lindau protein (pVHL), cullin E3 ligases, and BRCA2. We describe the NMR solution structure of the DUSP domain of human USP15, recently implicated in COP9 (constitutive photomorphogenic gene 9)-signalosome regulation. Its tripod-like structure consists of a 3-fold alpha-helical bundle supporting a triple-stranded anti-parallel beta-sheet. The DUSP domain displays a novel fold, an alpha/beta tripod (AB3). DUSP domain surface properties and previously described work suggest a potential role in protein/protein interaction or substrate recognition.

Amino Acid Sequence↗

PubMed2005

The structure of bypass of forespore C, an intercompartmental signaling factor during sporulation in Bacillus.

Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase sigma-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing sigma(K), (Esigma(K)). sigma(K) is synthesized as an inactive zymogen, pro-sigma(K), which is activated by proteolytic cleavage. Processing of pro-sigma(K) is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-sigma(K) processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-sigma(K) processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded beta-sheet onto one face of which an alpha-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded beta-sheet and three alpha-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the sigma(K) checkpoint are discussed in the light of sequence and structure comparisons.

Amino Acid Motifs↗

PubMed2005

RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank.

State-of-the-art methods based on CNS and CYANA were used to recalculate the nuclear magnetic resonance (NMR) solution structures of 500+ proteins for which coordinates and NMR restraints are available from the Protein Data Bank. Curated restraints were obtained from the BioMagResBank FRED database. Although the original NMR structures were determined by various methods, they all were recalculated by CNS and CYANA and refined subsequently by restrained molecular dynamics (CNS) in a hydrated environment. We present an extensive analysis of the results, in terms of various quality indicators generated by PROCHECK and WHAT_CHECK. On average, the quality indicators for packing and Ramachandran appearance moved one standard deviation closer to the mean of the reference database. The structural quality of the recalculated structures is discussed in relation to various parameters, including number of restraints per residue, NOE completeness and positional root mean square deviation (RMSD). Correlations between pairs of these quality indicators were generally low; for example, there is a weak correlation between the number of restraints per residue and the Ramachandran appearance according to WHAT_CHECK (r = 0.31). The set of recalculated coordinates constitutes a unified database of protein structures in which potential user- and software-dependent biases have been kept as small as possible. The database can be used by the structural biology community for further development of calculation protocols, validation tools, structure-based statistical approaches and modeling. The RECOORD database of recalculated structures is publicly available from http://www.ebi.ac.uk/msd/recoord.

Databases, Protein↗

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

BioMagResBank databases DOCR and FRED containing converted and filtered sets of experimental NMR restraints and coordinates from over 500 protein PDB structures.

We present two new databases of NMR-derived distance and dihedral angle restraints: the Database Of Converted Restraints (DOCR) and the Filtered Restraints Database (FRED). These databases currently correspond to 545 proteins with NMR structures deposited in the Protein Databank (PDB). The criteria for inclusion were that these should be unique, monomeric proteins with author-provided experimental NMR data and coordinates available from the PDB capable of being parsed and prepared in a consistent manner. The Wattos program was used to parse the files, and the CcpNmr FormatConverter program was used to prepare them semi-automatically. New modules, including a new implementation of Aqua in the BioMagResBank (BMRB) software Wattos were used to analyze the sets of distance restraints (DRs) for inconsistencies, redundancies, NOE completeness, classification and violations with respect to the original coordinates. Restraints that could not be associated with a known nomenclature were flagged. The coordinates of hydrogen atoms were recalculated from the positions of heavy atoms to allow for a full restraint analysis. The DOCR database contains restraint and coordinate data that is made consistent with each other and with IUPAC conventions. The FRED database is based on the DOCR data but is filtered for use by test calculation protocols and longitudinal analyses and validations. These two databases are available from websites of the BMRB and the Macromolecular Structure Database (MSD) in various formats: NMR-STAR, CCPN XML, and in formats suitable for direct use in the software packages CNS and CYANA.

Databases, Protein↗

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↗

PubMed2005

Extended flip-back schemes for sensitivity enhancement in multidimensional HSQC-type out-and-back experiments.

In many NMR experiments, only polarisation of a limited sub-set of all protons is converted into observable coherence. As recently shown by the "longitudinal" TROSY implementation (Pervushin et al. (2002) J. Am. Chem. Soc., 124, 12898-12902) and SOFAST-HMQC (Schanda and Brutscher (2005) J. Am. Chem. Soc., 127, 8014-8015), recovery of unused polarisation can be used indirectly and unspecifically to cool the proton lattice and, thus, accelerate re-equilibration for the selected proton subset. Here we illustrate transfer of this principle to HSQC-based multi-dimensional out-and-back experiments that exploit only polarisation of 15N-bound protons. The presented modifications to the pulse sequences can be implemented broadly and easily, extending standard flip-back of water polarisation to a much larger pool of protons that may comprise all non-15N-bound protons. The underlying orthogonal separation of H(N) polarisation (selected by the main transfer path) from unused H(u) polarisation (flipped-back on the recovery path) is thereby achieved through positive or negative selection by J-coupling, or using band-selective pulses. In practice, H(u) polarisation recovery degrades mostly through cumulative pulse imperfections and transverse relaxation; we present, however, strategies to substantially minimise such losses particularly during interim proton decoupling. Depending on the protein's relaxation properties and the extended flip-back scheme employed, we recovered up to 60% H(u) equilibrium polarisation. The concomitant cooling of the proton lattice afforded substantial gains of more than 40%, relative to the water-only flip-back version, in the fast pulsing regime with re-equilibration delays tau much shorter than optimal (tau(opt) = 1.25 x T1(H(N))). These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays tau(opt) can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.

Magnetic Resonance Spectroscopy↗

PubMed2005

NMR analysis of protein interactions.

Recent technological advances in NMR spectroscopy have alleviated the size limitations for the determination of biomolecular structures in solution. At the same time, novel NMR parameters such as residual dipolar couplings are providing greater accuracy. As this review shows, the structures of protein-protein and protein-nucleic acid complexes up to 50 kDa can now be accurately determined. Although de novo structure determination still requires considerable effort, information on interaction surfaces from chemical shift perturbations is much easier to obtain. Advances in modelling and data-driven docking procedures allow this information to be used for determining approximate structures of biomolecular complexes. As a result, a wealth of information has become available on the way in which proteins interact with other biomolecules. Of particular interest is the fact that these NMR-based methods can be applied to weak and transient protein-protein complexes that are difficult to study by other structural methods.

DNA↗

PubMed2005

The solution structure of a transient photoreceptor intermediate: Delta25 photoactive yellow protein.

The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

Bacterial Proteins↗

PubMed2005

The structure of the human ERCC1/XPF interaction domains reveals a complementary role for the two proteins in nucleotide excision repair.

The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.

Amino Acid Sequence↗

PubMed2004

The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics.

The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.

Anti-Bacterial Agents↗