Using chemical shift perturbation to characterise ligand binding.
TL;DR: By use of quantitative calculations of ligand-induced chemical shift changes, it is becoming possible to determine not just the position but also the orientation of ligands, and in particular the measurement of (13)CH3 signals.
About: This article is published in Progress in Nuclear Magnetic Resonance Spectroscopy.The article was published on 2013-08-01. It has received 1085 citations till now. The article focuses on the topics: Chemical shift & Ligand (biochemistry).
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TL;DR: The results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquit in and function as reversibly associated proteasomal Ubiquitin receptors.
Abstract: Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1. A site ( T1: ) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like ( UBL: ) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site ( T2: ) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.
237 citations
Journal Article•
206 citations
TL;DR: Methyl-TROSY (transverse relaxation optimized spectroscopy) NMR is highlighted, which enables the study of such large systems, and it is shown how this emerging technique contributes to an understanding of cellular function and the role of molecular plasticity in regulating an array of biochemical activities.
Abstract: Large macromolecular assemblies, so-called molecular machines, are critical to ensuring proper cellular function. Understanding how proper function is achieved at the atomic level is crucial to advancing multiple avenues of biomedical research. Biophysical studies often include X-ray diffraction and cryo-electron microscopy, providing detailed structural descriptions of these machines. However, their inherent flexibility has complicated an understanding of the relation between structure and function. Solution NMR spectroscopy is well suited to the study of such dynamic complexes, and continued developments have increased size boundaries; insights into function have been obtained for complexes with masses as large as 1 MDa. We highlight methyl-TROSY (transverse relaxation optimized spectroscopy) NMR, which enables the study of such large systems, and include examples of applications to several cellular machines. We show how this emerging technique contributes to an understanding of cellular function and the role of molecular plasticity in regulating an array of biochemical activities.
188 citations
TL;DR: It is demonstrated that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.
Abstract: The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014 This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells
165 citations
TL;DR: The molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis is described and how the LysM module recognizes the GlcNAc-X-Glc NAc motif present in polysaccharides across kingdoms is revealed.
Abstract: Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM-peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.
163 citations
References
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Book•
15 Sep 1998
TL;DR: The three-dimensional structure of proteins chemical catalysis the basic equations of enzyme kinetics measurement and magnitude of enzymatic rate constants the pH dependence of enzyme catalysis practical kinetics detection of intermediaries in reactions by kinetics stereochemistry of enzymes reactions active-site-directed and enzyme-activated irreversible inhibitors - affinity labels and suicide inhibitors conformational change, allosteric regulation, motors and work forces between molecules, and enzymesubstrate binding energies enzyme-substrate complementarity and the use of binding energy in catalysis specificity and editing mechanisms recombinant DNA technology case studies of enzyme
Abstract: The three-dimensional structure of proteins chemical catalysis the basic equations of enzyme kinetics measurement and magnitude of enzymatic rate constants the pH dependence of enzyme catalysis practical kinetics detection of intermediaries in reactions by kinetics stereochemistry of enzymic reactions active-site-directed and enzyme-activated irreversible inhibitors - affinity labels and suicide inhibitors conformational change, allosteric regulation, motors and work forces between molecules, and enzyme-substrate binding energies enzyme-substrate complementarity and the use of binding energy in catalysis specificity and editing mechanisms recombinant DNA technology case studies of enzyme structure and mechanism protein engineering protein stability kinetics of protein folding folding pathways and energy landscapes.
2,677 citations
TL;DR: An approach called HADDOCK (High Ambiguity Driven protein-protein Docking) that makes use of biochemical and/or biophysical interaction data such as chemical shift perturbation data resulting from NMR titration experiments or mutagenesis data to drive the docking process.
Abstract: The structure determination of protein-protein complexes is a rather tedious and lengthy process, by both NMR and X-ray crystallography. Several methods based on docking to study protein complexes have also been well developed over the past few years. Most of these approaches are not driven by experimental data but are based on a combination of energetics and shape complementarity. Here, we present an approach called HADDOCK (High Ambiguity Driven protein-protein Docking) that makes use of biochemical and/or biophysical interaction data such as chemical shift perturbation data resulting from NMR titration experiments or mutagenesis data. This information is introduced as Ambiguous Interaction Restraints (AIRs) to drive the docking process. An AIR is defined as an ambiguous distance between all residues shown to be involved in the interaction. The accuracy of our approach is demonstrated with three molecular complexes. For two of these complexes, for which both the complex and the free protein structures have been solved, NMR titration data were available. Mutagenesis data were used in the last example. In all cases, the best structures generated by HADDOCK, that is, the structures with the lowest intermolecular energies, were the closest to the published structure of the respective complexes (within 2.0 A backbone RMSD).
2,616 citations
"Using chemical shift perturbation t..." refers methods in this paper
...Clore and Schwieters described a very similar method to HADDOCK [90]....
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...A recent version of HADDOCK also uses this approach [37]: it uses shifts in the standard qualitative way first, to locate the binding site, but then uses a more complete calculation (using the empirical calculation carried out by ShiftX [104]) to optimise the poses....
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...Similar programs have been described by others but have not achieved the popularity of HADDOCK....
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...HADDOCK also defines passive residues, which are surface residues that either have a smaller shift change than the active residues or are close to the active residues....
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...The most popular program using this method is HADDOCK [85]....
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TL;DR: The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.
Abstract: Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.
2,262 citations
"Using chemical shift perturbation t..." refers methods in this paper
...With TROSY and perdeuterated protein [2] one can observe proteins of several hundred kDa, so that (for a well-behaved protein, and with a bit of effort) one can acquire CSP data on most targets of interest....
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TL;DR: A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands and appears particularly useful in target-directed drug research.
Abstract: A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands. The approach is called "SAR by NMR" because structure-activity relationships (SAR) are obtained from NMR. With this technique, compounds with nanomolar affinities for the FK506 binding protein were rapidly discovered by tethering two ligands with micromolar affinities. The method reduces the amount of chemical synthesis and time required for the discovery of high-affinity ligands and appears particularly useful in target-directed drug research.
1,971 citations
TL;DR: An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships.
1,862 citations
"Using chemical shift perturbation t..." refers background in this paper
...For example, it has been noted that the spread of chemical shifts for glycine is different from other amino acids [54], justifying a = 0....
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