Resonance assignment of methionine methyl groups and χ3angular information from long-range proton—carbon and carbon—carbon J correlation in a calmodulin—peptide complex

Summary Several simple 3D experiments are used to provide J correlations between methionine C methyl carbons and either the CH₂ protons or C and C. The intensity of the J correlations provides information on the size of the three-bond J couplings and thereby on the ³ torsion angle. In addition, a simple 3D version of the HMBC experiment provides a sensitive link between the CH₃ methyl protons and C. The methods are demonstrated for a 20 kDa complex between calmodulin and a 26-residue peptide fragment of skeletal muscle myosin light chain kinase.     

The EH1 domain of Eps15 is structurally classified as a member of the S100 subclass of EF-hand-containing proteins.

The Eps15 homology (EH) domain is a protein-protein interaction module that binds to proteins containing the asparagine-proline-phenylalanine (NPF) or tryptophan/phenylalanine-tryptophan (W/FW) motif. EH domain-containing proteins serve important roles in signaling and processes connected to transport, protein sorting, and organization of subcellular structure. Here, we report the solution structure of the apo form of the EH1 domain of mouse Eps15, as determined by high-resolution multidimensional heteronuclear NMR spectroscopy. The polypeptide folds into six alpha-helices and a short antiparallel beta-sheet. Additionally, it contains a long, structured, topologically unique C-terminal loop. Helices 2-5 form two EF-hand motifs. Structural similarity and Ca(2+) binding properties lead to classification of the EH1 domain as a member of the S100 subclass of EF-hand-containing proteins, albeit with a unique set of interhelical angles. Binding studies using an eight-residue NPF-containing peptide derived from RAB, the cellular cofactor of the HIV Rev protein, show a hydrophobic peptide-binding pocket formed by conserved tryptophan and leucine residues.     

How to choose templates for modeling of protein complexes: Insights from benchmarking template-based docking

Comparative docking is based on experimentally determined structures of protein-protein complexes (templates), following the paradigm that proteins with similar sequences and/or structures form similar complexes. Modeling utilizing structure similarity of target monomers to template complexes significantly expands structural coverage of the interactome. Template-based docking by structure alignment can be performed for the entire structures or by aligning targets to the bound interfaces of the experimentally determined complexes. Systematic benchmarking of docking protocols based on full and interface structure alignment showed that both protocols perform similarly, with top 1 docking success rate 26%. However, in terms of the models' quality, the interface-based docking performed marginally better. The interface-based docking is preferable when one would suspect a significant conformational change in the full protein structure upon binding, for example, a rearrangement of the domains in multidomain proteins. Importantly, if the same structure is selected as the top template by both full and interface alignment, the docking success rate increases 2-fold for both top 1 and top 10 predictions. Matching structural annotations of the target and template proteins for template detection, as a computationally less expensive alternative to structural alignment, did not improve the docking performance. Sophisticated remote sequence homology detection added templates to the pool of those identified by structure-based alignment, suggesting that for practical docking, the combination of the structure alignment protocols and the remote sequence homology detection may be useful in order to avoid potential flaws in generation of the structural templates library.     

Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry gate for active Ca(2+) reabsorption in the kidney. Ca(2+) influx through TRPV5 induces rapid channel inactivation, preventing excessive Ca(2+) influx. This inactivation is mediated by the last ～30 residues of the carboxy (C) terminus of the channel. Since the Ca(2+)-sensing protein calmodulin has been implicated in Ca(2+)-dependent regulation of several TRP channels, the potential role of calmodulin in TRPV5 function was investigated. High-resolution nuclear magnetic resonance (NMR) spectroscopy revealed a Ca(2+)-dependent interaction between calmodulin and a C-terminal fragment of TRPV5 (residues 696 to 729) in which one calmodulin binds two TRPV5 C termini. The TRPV5 residues involved in calmodulin binding were mutated to study the functional consequence of releasing calmodulin from the C terminus. The point mutants TRPV5-W702A and TRPV5-R706E, lacking calmodulin binding, displayed a strongly diminished Ca(2+)-dependent inactivation compared to wild-type TRPV5, as demonstrated by patch clamp analysis. Finally, parathyroid hormone (PTH) induced protein kinase A (PKA)-dependent phosphorylation of residue T709, which diminished calmodulin binding to TRPV5 and thereby enhanced channel open probability. The TRPV5-W702A mutant exhibited a significantly increased channel open probability and was not further stimulated by PTH. Thus, calmodulin negatively modulates TRPV5 activity, which is reversed by PTH-mediated channel phosphorylation.     

Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis

Proton NMR spectra of a dimeric phospholipase A₂ from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A₂ from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.     

Application of 2D and 3D NMR experiments to the conformational study of a diantennary oligosaccharide

Summary By the application of homonuclear 3D NOE-HOHAHA and heteronuclear 3D HMQC-NOE experiments in studies of complex oligosaccharides. NOEs can be investigated which are hidden in conventional 2D NOE spectra. In the 3D NOE-HOHAHA spectrum ₃ cross sections were considered to be the most suitable for assignment of NOEs. Alternatively, these cross sections could be measured separately in selective 2D HOHAHA-NOE spectroscopy. The advantages and limitations of the 2D alternative are compared with those of the 3D NOE-HOHAHA approach. In 3D HMQC-NOE spectroscopy the larger chemical shift displacement of the carbon spectrum with respect to the proton spectrum can be used to unmask NOEs hidden in the bulk region. If the extra proton dimension is not needed, 2D HMQC-NOE is a good alternative.The suitability of 2D and 3D NOE-HOHAHA and HMQC-NOE experiments for the estimation of proton-proton distances is demonstrated by comparing the results of these experiments on a diantennary asparagine-linked oligosaccharide with those of a conventional 2D NOE experiment. NOEs identified in the 2D and 3D NOE-HOHAHA as well as HMQC-NOE experiments, so far not identified or not quantified in 2D NOE experiments, are discussed in relation to each glycosidic linkage. The flexibility of the Man(1-3)Man linkage is demonstrated, confirming the existence of an ensemble of conformations for this linkage.