Double electron–electron resonance (DEER) distance measurements of a protein complex tagged with two Gd3+ chelates developed for rigid positioning of the metal ion are shown to deliver outstandingly ...accurate distance measurements in the 6 nm range. The accuracy was assessed by comparison with modeled distance distributions based on the three-dimensional molecular structures of the protein and the tag and further comparison with paramagnetic NMR data. The close agreement between the predicted and experimentally measured distances opens new possibilities for investigating the structure of biomolecular assemblies. As an example, we show that the dimer interface of rat ERp29 in solution is the same as that determined previously for human ERp29 in the single crystal.
n-Alkyl-poly(ethylene glycol)/n-alkyl alcohol and glucopone/n-hexanol mixtures are shown to form dilute liquid crystalline phases in aqueous solution which are suitable for partial alignment of ...biological macromolecules in a magnetic field. The constituent compounds are commercially available and inexpensive. The poly(ethylene glycol)-based systems are uncharged and thus insensitive with respect to pH, little sensitive with respect to salt, and tolerant against high protein concentrations. They are demonstrated to be suitable for measurements of residual dipolar couplings in proteins, DNA, and a protein/DNA complex. The stability range of the lamellar phase with respect to temperature was explored for different mixtures and guidelines are provided for the use of these lyotropic systems in NMR spectroscopy.
The paramagnetism of lanthanide ions offers outstanding opportunities for fast determinations of the three-dimensional (3D) structures of protein−ligand complexes by nuclear magnetic resonance (NMR) ...spectroscopy. It is shown how the combination of pseudocontact shifts (PCSs) induced by a site-specifically bound lanthanide ion and prior knowledge of the 3D structure of the lanthanide-labeled protein can be used to achieve (i) rapid assignments of NMR spectra, (ii) structure determinations of protein−protein complexes, and (iii) identification of the binding mode of low-molecular weight compounds in complexes with proteins. Strategies for site-specific incorporation of lanthanide ions into proteins are summarized.
Structure-guided drug design relies on detailed structural knowledge of protein–ligand complexes, but crystallization of cocomplexes is not always possible. Here we present a sensitive nuclear ...magnetic resonance (NMR) approach to determine the binding mode of tightly binding lead compounds in complex with difficult target proteins. In contrast to established NMR methods, it does not depend on rapid exchange between bound and free ligand or on stable isotope labeling, relying instead on a tert-butyl group as a chemical label. tert-Butyl groups are found in numerous protein ligands and deliver an exceptionally narrow and tall 1H NMR signal. We show that a tert-butyl group also produces outstandingly intense intra- and intermolecular NOESY cross-peaks. These enable measurements of pseudocontact shifts generated by lanthanide tags attached to the protein, which in turn allows positioning of the ligand on the protein. Once the ligand has been located, assignments of intermolecular NOEs become possible even without prior resonance assignments of protein side chains. The approach is demonstrated with the dengue virus NS2B-NS3 protease in complex with a high-affinity ligand containing a tert-butyl group.
The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully ...active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro (“open state”), necessitating a large structural change to produce the “closed state” thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of 15N-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd3+ complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned 15N-HSQC spectra.
Pulse electron paramagnetic resonance measurements of long-range (nm scale) distances between spin labels site-specifically attached to biomacromolecules have proven highly effective in structural ...studies. The most commonly used spin labels are stable nitroxide radicals, and measurements are usually carried out at X-band frequencies (∼9.5 GHz, 0.35 T). Higher magnetic fields open new possibilities for distance measurements with increased sensitivity using alternative spin labels containing half-integer high-spin metal ions. Here we demonstrate W-band (95 GHz) pulse double electron–electron resonance (DEER) distance measurements in a protein labeled with two Mn2+-EDTA tags. The distance distribution obtained is in excellent agreement with model calculations based on the known solution NMR structure. Thus, site-specific labeling with Mn2+ tags opens a highly promising approach to nanometer distance measurements in biological macromolecules.
Capturing conformational changes in proteins or protein-protein complexes is a challenge for both experimentalists and computational biologists. Solution nuclear magnetic resonance (NMR) is unique in ...that it permits structural studies of proteins under greatly varying conditions, and thus allows us to monitor induced structural changes. Paramagnetic effects are increasingly used to study protein structures as they give ready access to rich structural information of orientation and long-range distance restraints from the NMR signals of backbone amides, and reliable methods have become available to tag proteins with paramagnetic metal ions site-specifically and at multiple sites. In this study, we show how sparse pseudocontact shift (PCS) data can be used to computationally model conformational states in a protein system, by first identifying core structural elements that are not affected by the environmental change, and then computationally completing the remaining structure based on experimental restraints from PCS. The approach is demonstrated on a 27 kDa two-domain NS2B-NS3 protease system of the dengue virus serotype 2, for which distinct closed and open conformational states have been observed in crystal structures. By changing the input PCS data, the observed conformational states in the dengue virus protease are reproduced without modifying the computational procedure. This data driven Rosetta protocol enables identification of conformational states of a protein system, which are otherwise difficult to obtain either experimentally or computationally.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three‐dimensional (3D) structure of an unstable, ...low‐abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site‐specific cysteine‐reactive paramagnetic tag that does not react with the active‐site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non‐equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.
Now you see it: 3D structures of low‐abundance transient enzyme intermediates can be determined from pseudocontact shifts (PCSs) measured by NMR spectroscopy. The method is demonstrated with the unstable thioester intermediate formed between Staphylococcus aureus sortase A and the peptide substrate.
Site-specific attachment of paramagnetic lanthanide ions to a protein generates pseudocontact shifts (PCS) in the nuclear magnetic resonance (NMR) spectra of the protein that are easily measured as ...changes in chemical shifts. By labeling the protein with lanthanide tags at four different sites, PCSs are observed for most amide protons and accurate information is obtained about their coordinates in three-dimensional space. The approach is demonstrated with the chaperone ERp29, for which large differences have been reported between X-ray and NMR structures of the C-terminal domain, ERp29-C. The results unambiguously show that the structure of rat ERp29-C in solution is similar to the crystal structure of human ERp29-C. PCSs of backbone amides were the only structural restraints required. Because these can be measured for more dilute protein solutions than other NMR restraints, the approach greatly widens the range of proteins amenable to structural studies in solution.
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•Four lanthanide tags allow determining atom positions in 3D space•NMR method for 3D fold determinations of proteins in solution•More sensitive than conventional NMR methods•Sufficient to measure restraints for backbone amides
Yagi et al. describe a method for protein structure determination in solution by NMR spectroscopy. The method uses only pseudocontact shifts of backbone amide protons generated by lanthanide ions attached at different sites. The method produced an improved 3D structure of the C-terminal domain of rat ERp29.
The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of ...selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.
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