The Conformation of Coenzyme A Bound to Chloramphenicol Acetyltransferase Determined by Transferred NOE Experiments

Barsukov, Igor; Lian, Lu‐Yun; Ellis, Jacqueline; Sze, KH; Shaw, William V.; Roberts, G. C. K.

doi:10.1006/jmbi.1996.0534

Cited by 22 publications

(19 citation statements)

References 14 publications

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“…In these studies, binding of cCRF 30 -41 to the isolated ECD of human CRF 1 receptors was characterized by using a combination of NMR spectroscopy techniques (27)(28)(29). A model of cCRF 30 -41 was constructed while bound to this recombinant ECD using transferred nuclear Overhauser effects (trNOE), in which a sufficient off-rate of the peptide from the ECD allowed information about interproton distances to be derived from the bound state of the peptide (30,31).…”

mentioning

confidence: 99%

NMR Structural Characterization of a Minimal Peptide Antagonist Bound to the Extracellular Domain of the Corticotropin-releasing Factor1 Receptor

Mesleh¹,

Shirley²,

Heise³

et al. 2007

Journal of Biological Chemistry

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Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF 1 receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF 1 receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30 -41(cCRF 30 -41 ), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an ␣-helical conformation when bound to the ECD. Residues of cCRF 30 -41 comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met 38 and Ile 41 ) as well as two amide groups (Asn 34 and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of nonpeptide antagonists targeting the ECD of this receptor.Corticotropin-releasing factor 1 (CRF 1 ) 2 receptor plays a crucial modulatory role in the central stress response, making it an attractive target for the development of therapeutics for a variety of stress-related disorders, including anxiety and depression (1-5). The CRF 1 receptor is a class B G-protein-coupled receptor (GPCR) and is activated endogenously in mammals by the peptide agonists CRF (6) and the urocortins (7-9). Binding of the native ligands to the receptor promotes a conformational change that leads to G-protein activation inside the cell. Signaling is mediated primarily through the second messenger cAMP via G␣ s ; however, coupling of the receptor to G␣ i and G␣ q/11 has been observed (10, 11). The receptor is expressed both in the periphery, where pituitary activation underlies the major role of CRF activation of the hypothalamic pituitary adrenal axis during stress, and in brain, where extrahypothalamic CRF activates central receptors to mediate mood and affect (12).A two-site binding and activation model has been proposed for agonist binding to class B GPCRs (13). Recent pharmacological data suggest that activation of the CRF 1 receptor is initiated by the C terminus (ϳ30 amino acids) of the peptide ligand interacting with relatively moderate affinity to the extracellular N-terminal domain (ECD) of the CRF 1 receptor (14). This primary interaction has the effect of increasing the local concentration of the ligand and positions its N terminus (ϳ10 amino acids) in the optimal orientation...

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confidence: 99%

NMR Structural Characterization of a Minimal Peptide Antagonist Bound to the Extracellular Domain of the Corticotropin-releasing Factor1 Receptor

Mesleh¹,

Shirley²,

Heise³

et al. 2007

Journal of Biological Chemistry

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“…For example, in a study of coenzyme A binding to chloramphenicol acetyltransferase [104], the dissociation rates estimated by NMR were 2.5-3 times faster than those measured by stopped-flow fluorescence experiments, and the conformation of the bound ligand determined by transferred NOE experiments (see below) differed from that determined by X-ray crystallography. Both of these discrepancies could be accounted for by a model in which the complex exists in two states, the predominant one dissociating more slowly and having the conformation seen in the crystal and the other having a more compact conformation of coenzyme A, with shorter internuclear distances and hence having a predominant influence on the NOE experiment.…”

Section: Fast Exchangementioning

confidence: 99%

“…Spin diffusion through the ligand protons decreases reasonably rapidly as the fraction of bound ligand decreases so that, under the conditions of excess ligand which are commonly used, it is less of a problem. It is also possible to use a relaxation matrix approach to include all the dipolar interactions between all the spins in the ligand, thus taking into account spin diffusion [104,120,130]. The success of this approach of course requires that a large number of trNOEs can be measured, so that a large number of distance restraints define the relative positions of the ligand protons and the spin diffusion effects can be estimated.…”

Section: Spin Diffusionmentioning

confidence: 99%

“…Transferred ROESY experiments can sometimes help to identify spin diffusion effects [120], but the only general solution is to use perdeuterated protein (e.g. [104]). …”

Section: Spin Diffusionmentioning

confidence: 99%

“…This effect was first observed in the ternary complex of chloramphenicol and coenzyme A with chloramphenicol acetyltransferase [104] and has subsequently been observed in a number of other systems [134][135][136][137][138][139] and named the ILOE (interligand Overhauser effect). A theoretical analysis has been developed for the evaluation of interligand Overhauser effects in a ternary complex [140], providing guidance on the conditions which must be fulfilled for correct interpretation.…”

Section: Two Ligands Bound Simultaneouslymentioning

confidence: 99%

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Structural and Dynamic Information on Ligand Binding

Roberts¹

2011

Protein NMR Spectroscopy: Practical Techniques and Applications

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The function of the vast majority of proteins in the cell depends upon their noncovalent interactions with other molecules, ranging from enzyme-substrate interactions to the array of protein-protein and protein-nucleic acid interactions involved in, for example, the regulation of transcription. NMR has proved very valuable in the study of the interaction of proteins with both small and large molecules. In this chapter the focus will be on the basic principles of the study of ligand binding by NMR and on applications to small moleculeprotein interactions. Studies of macromolecular complexes are covered in Chapter 8. The use of NMR to study protein-ligand interactions is a vast area and, for reasons of space, many of the references herein are to reviews rather than to original papers; my apologies to those whose important contributions have not been cited directly.NMR can of course provide information on the structure of protein-ligand complexes, just as it can on the structure of the protein itself. However, the NMR spectrum is sensitive to both the equilibrium and the kinetics of the binding process. These effects can provide valuable information in themselves but, most importantly, they must be understood in order to interpret the spectrum correctly.

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The structural basis for substrate versatility of chloramphenicol acetyltransferase CAT_I

2012

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Novel antibiotics are needed to overcome the challenge of continually evolving bacterial resistance. This has led to a renewed interest in mechanistic studies of once popular antibiotics like chloramphenicol (CAM). Chloramphenicol acetyltransferases (CATs) are enzymes that covalently modify CAM, rendering it inactive against its target, the ribosome, and thereby causing resistance to CAM. Of the three major types of CAT (CATI‐III), the CAM‐specific CATIII has been studied extensively. Much less is known about another clinically important type, CATI. In addition to inactivating CAM and unlike CATIII, CATI confers resistance to a structurally distinct antibiotic, fusidic acid. The origin of the broader substrate specificity of CATI has not been fully elucidated. To understand the substrate binding features of CATI, its crystal structures in the unbound (apo) and CAM‐bound forms were determined. The analysis of these and previously determined CATI‐FA and CATIII‐CAM structures revealed interactions responsible for CATI binding to its substrates and clarified the broader substrate preference of CATI compared to that of CATIII.

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The Conformation of Coenzyme A Bound to Chloramphenicol Acetyltransferase Determined by Transferred NOE Experiments

Cited by 22 publications

References 14 publications

NMR Structural Characterization of a Minimal Peptide Antagonist Bound to the Extracellular Domain of the Corticotropin-releasing Factor1 Receptor

NMR Structural Characterization of a Minimal Peptide Antagonist Bound to the Extracellular Domain of the Corticotropin-releasing Factor1 Receptor

Structural and Dynamic Information on Ligand Binding

The structural basis for substrate versatility of chloramphenicol acetyltransferase CAT_I

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The Conformation of Coenzyme A Bound to Chloramphenicol Acetyltransferase Determined by Transferred NOE Experiments

Cited by 22 publications

References 14 publications

NMR Structural Characterization of a Minimal Peptide Antagonist Bound to the Extracellular Domain of the Corticotropin-releasing Factor1 Receptor

NMR Structural Characterization of a Minimal Peptide Antagonist Bound to the Extracellular Domain of the Corticotropin-releasing Factor1 Receptor

Structural and Dynamic Information on Ligand Binding

The structural basis for substrate versatility of chloramphenicol acetyltransferase CATI

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The structural basis for substrate versatility of chloramphenicol acetyltransferase CAT_I