High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment

Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 Å from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions

Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein–protein complex structure determination

Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein–protein and protein–ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination

Stability and kinetics of G-quadruplex structures

In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence

Cooperation between a T Domain and a Minimal C‐Terminal Docking Domain to Enable Specific Assembly in a Multiprotein NRPS

Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains ((N/C)DDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of "designer" NRPS from proteins that normally do not interact. The multiprotein "peptide-antimicrobial-Xenorhabdus" (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small (C)DD of PaxA cooperates with its preceding thiolation (T-1) domain to bind the (DD)-D-N of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems

Xentrivalpeptides A-Q: depsipeptide diversification in Xenorhabdus

Item does not contain fulltextSeventeen depsipeptides, xentrivalpeptides A-Q (1-17), have been identified from an entomopathogenic Xenorhabdus sp. Whereas the structure of xentrivalpeptide A (1) was determined after its isolation by NMR spectroscopy and the advanced Marfey's method, the structures of all other derivatives were determined using a combination of stable isotope labeling and detailed MS analysis

Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.

It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements

Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution

Structural transitions of RNA between alternate conformations with similar stabilities are associated with important aspects of cellular function. Few techniques presently exist that are capable of monitoring such transitions and thereby provide insight into RNA dynamics and function at atomic resolution. Riboswitches are found in the 5′-UTR of mRNA and control gene expression through structural transitions after ligand recognition. A time-resolved NMR strategy was established in conjunction with laser-triggered release of the ligand from a photocaged derivative in situ to monitor the hypoxanthine-induced folding of the guanine-sensing riboswitch aptamer domain of the Bacillus subtilis xpt-pbuX operon at atomic resolution. Combining selective isotope labeling of the RNA with NMR filter techniques resulted in significant spectral resolution and allowed kinetic analysis of the buildup rates for individual nucleotides in real time. Three distinct kinetic steps associated with the ligand-induced folding were delineated. After initial complex encounter the ligand-binding pocket is formed and results in subsequent stabilization of a remote long-range loop–loop interaction. Incorporation of NMR data into experimentally restrained molecular dynamics simulations provided insight into the RNA structural ensembles involved during the conformational transition