NMR detection of protein 15N spins near paramagnetic lanthanide ions.
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Ions↗
Biomedical subjects
Gottfried Otting
Publications and source records attributed to Gottfried Otting.
Assignment of paramagnetic (15)N-HSQC spectra by heteronuclear exchange spectroscopy.
Paramagnetic metal ions in proteins provide a rich source of structural information, but the resonance assignments required to extract the information can be challenging. Here we demonstrate that paramagnetically shifted (15)N-HSQC cross-peaks can be assigned using N(Z)-exchange spectroscopy under conditions in which the paramagnetic form of the protein is in dynamic equilibrium with its diamagnetic form. Even slow exchange of specifically bound metal ions may be detected within the long lifetime of (15)N longitudinal magnetization of large proteins at high magnetic fields. Alternatively, the exchange can be accelerated using an excess of metal ions. In the resulting exchange spectra, paramagnetic (15)N resonances become visible for residues that are not directly observed in a conventional (15)N-HSQC spectrum due to paramagnetic (1)H(N) broadening. The experiments are illustrated by the 30 kDa lanthanide-binding epsilon186/theta complex of DNA polymerase III in the presence of sub-stoichiometric amounts of Dy(3+) or a mixture of Dy(3+) and La(3+).
Algorithms↗
Structure determination of protein-ligand complexes by transferred paramagnetic shifts.
Rational drug design depends on the knowledge of the three-dimensional (3D) structure of complexes between proteins and lead compounds of low molecular weight. A novel nuclear magnetic resonance (NMR) spectroscopy strategy based on the paramagnetic effects from lanthanide ions allows the rapid determination of the 3D structure of a small ligand molecule bound to its protein target in solution and, simultaneously, its location and orientation with respect to the protein. The method relies on the presence of a lanthanide ion in the protein target and on fast exchange between bound and free ligand. The binding affinity of the ligand and the paramagnetic effects experienced in the bound state are derived from concentration-dependent (1)H and (13)C spectra of the ligand at natural isotopic abundance. Combined with prior knowledge of the crystal or solution structure of the protein and of the magnetic susceptibility tensor of the lanthanide ion, the paramagnetic data define the location and orientation of the bound ligand molecule with respect to the protein from simple 1D NMR spectra. The method was verified with the ternary 30 kDa complex between the lanthanide-labeled N-terminal domain of the epsilon exonuclease subunit from the Escherichia coli DNA polymerase III, the subunit theta, and thymidine. The binding mode of thymidine was found to be very similar to that of thymidine monophosphate present in the crystal structure.
DNA Polymerase III↗
Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase.
DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1-171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1-171 of the E. coli DnaB helicase with significant affinity.
Amino Acid Sequence↗
N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes.
[(15)N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [(15)N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [(15)N]-HSQC spectra by amino acid type selective [(15)N]-labelling. The samples can be measured without chromatographic protein purification, dilution of isotopes by transaminase activities are suppressed, and a combinatorial isotope labelling scheme can be adopted that combines reduced spectral overlap with a minimum number of samples for the identification of all [(15)N]-HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking [(15)N]-HSQC cross-peaks after titration with unlabelled ligand molecules or macromolecular binding partners. In particular, combinatorial isotope labelling can provide complete cross-peak identification by amino acid type in 24 h, including protein production and NMR measurement.
Cell-Free System↗
Efficient chi-tensor determination and NH assignment of paramagnetic proteins.
Anisotropic magnetic susceptibility tensors chi of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the chi-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D 15N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable chi-tensor anisotropy parameters from 2D spectra of uniformly 15N-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit epsilon from E. coli DNA polymerase III in complex with the subunit theta and La3+ in its diamagnetic and Dy3+, Tb3+, and Er3+ in its paramagnetic form.
Algorithms↗
Second Kunitz-type protease inhibitor domain of the human WFIKKN1 protein.
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Amino Acid Sequence↗
Lanthanide labeling offers fast NMR approach to 3D structure determinations of protein-protein complexes.
A novel nuclear magnetic resonance (NMR) strategy based on labeling with lanthanides achieves rapid determinations of accurate three-dimensional (3D) structures of protein-protein complexes. The method employs pseudocontact shifts (PCS) induced by a site-specifically bound lanthanide ion to anchor the coordinate system of the magnetic susceptibility tensor in the molecular frames of the two molecules. Simple superposition of the tensors detected in the two protein molecules brings them together in a 3D model of the protein-protein complex. The method is demonstrated with the 30 kDa complex between two subunits of Escherichia coli polymerase III, comprising the N-terminal domain of the exonuclease subunit epsilon and the subunit theta. The 3D structures of the individual molecules were docked based on a limited number of PCS observed in 2D 15N-heteronuclear single quantum coherence spectra. Degeneracies in the mutual orientation of the protein structures were resolved by the use of two different lanthanide ions, Dy3+ and Er3+.
DNA Polymerase III↗
NMR structure of the WIF domain of the human Wnt-inhibitory factor-1.
The human Wnt-binding protein Wnt-inhibitory factor-1 (WIF-1) comprises an N-terminal WIF module followed by five EGF-like repeats. Here we report the three-dimensional structure of the WIF domain of WIF-1 determined by NMR spectroscopy. The fold consists of an eight-stranded beta-sandwich reminiscent of the immunoglobulin fold. Residual detergent (Brij-35) used in the refolding protocol was found to bind tightly to the WIF domain. The binding site was identified by intermolecular nuclear Overhauser effects observed between the WIF domain and the alkyl chain of the detergent. The results point to a possible role of WIF domains as a recognition motif of Wnt and Drosophila Hedgehog proteins that are activated by palmitoylation.
Adaptor Proteins, Signal Transducing↗
Site-specific labelling of proteins with a rigid lanthanide-binding tag.
This paper describes a generic method for the site-specific attachment of lanthanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Escherichia coli arginine repressor. Complexes with Y(3+), Tb(3+), Dy(3+), Ho(3+), Er(3+), Tm(3+) and Yb(3+) ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisotropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.
Amino Acid Sequence↗
Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling.
The efficiency of cell-free protein synthesis combined with combinatorial selective 15N-labelling provides a method for the rapid assignment of 15N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five 15N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the tau subunit of the Escherichia coli DNA polymerase III holoenzyme, tauC16 and tauC14. Since each of the five 15N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All 15N-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in tauC16 vs. those in tauC14, and the analysis of the structure and mobility of the C-terminal residues present in tauC16 but not in tauC14.
Amino Acid Sequence↗
Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit.
The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.
Catalytic Domain↗
Weak alignment of paramagnetic proteins warrants correction for residual CSA effects in measurements of pseudocontact shifts.
Paramagnetic metal ions can induce molecular alignment with respect to the magnetic field. This alignment generates residual anisotropic chemical shifts (RACS) due to nonisotropic averaging over the molecular orientations. Using a 30 kDa protein-protein complex, the RACS effects are shown to be significant for heteronuclear spins with large chemical shift anisotropies, lanthanide ions with large anisotropic magnetic susceptibility tensors, and measurements at high magnetic field. Therefore, RACS must be taken into account when pseudocontact shifts are measured by comparison of chemical shifts observed between complexes with paramagnetic and diamagnetic lanthanide ions. The results are of particular importance when different pseudocontact shifts measured for the 1HN, 15N, and 13C' spins of a peptide group are used to restrain its orientation with respect to the electronic magnetic susceptibility tensor in structure calculations.
Anisotropy↗
Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy.
Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, alpha3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.
Bacillus subtilis↗
Stabilization of native protein fold by intein-mediated covalent cyclization.
A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.
Adenosine Triphosphatases↗
Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC.
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.
Amino Acid Sequence↗
Crystal and solution structures of the helicase-binding domain of Escherichia coli primase.
During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase. In Escherichia coli, this occurs by transient interaction of primase with the helicase. Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase is responsible for interaction with DnaB6. Determination of the 2.8-angstroms crystal structure of the C-terminal domain of primase revealed an asymmetric dimer. The monomers have an N-terminal helix bundle similar to the N-terminal domain of DnaB, followed by a long helix that connects to a C-terminal helix hairpin. The connecting helix is interrupted differently in the two monomers. Solution studies using NMR showed that an equilibrium exists between a monomeric species with an intact, extended but naked, connecting helix and a dimer in which this helix is interrupted in the same way as in one of the crystal conformers. The other conformer is not significantly populated in solution, and its presence in the crystal is due largely to crystal packing forces. It is proposed that the connecting helix contributes necessary structural flexibility in the primase-helicase complex at replication forks.
Amino Acid Sequence↗
Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein-protein complexes even if one of the protein components is prone to self-aggregation and precipitation. Combined with selective isotope-labeling, this allows the rapid analysis of protein-protein interactions with few 15N-HSQC spectra. The concept is demonstrated with binary and ternary complexes between the chi, psi and gamma subunits of Escherichia coli DNA polymerase III: nascent, selectively 15N-labeled psi produced in the presence of chi resulted in a soluble, correctly folded chi-psi complex, whereas psi alone precipitated irrespective of whether gamma was present or not. The 15N-HSQC spectra showed that the N-terminal segment of psi is mobile in the chi-psi complex, yet important for its binding to gamma. The sample preparation was greatly enhanced by an autoinduction strategy, where the T7 RNA polymerase needed for transcription of a gene in a T7-promoter vector was produced in situ.
Cell-Free System↗