Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)


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As noted earlier for the individual chains, in one of the replicates involving the EMPbound system Fig. RMSD values for the sEPOR 2 dimer calculated over the backbone atoms after first performing a least-squares fit onto the backbone atoms of the chain A. Green , red , blue , orange , gray Independent simulations 1—5, respectively.

Each sEPOR chain was represented as a plane as described above. Black dots Values of the initial conformations. In the fifth simulation gray dots in Fig. In the apo dimer Fig. In the simulations, D 2 d sampled a range of values 1. In addition, sEPOR 2 bound to a range of other agonistic and antagonistic ligands was also investigated. Green , red , blue Simulations 1—3, respectively.

In contrast, the value of D 2 d varied significantly depending on the origin of the starting structure.

Likewise, D 2 d varied between 2. The EPOR is a primary regulator of erythropoiesis but has also been recently identified in neurons where it helps protect against brain injury Its mechanism of activation, how the binding of a ligand on the extracellular domains is transmitted through the plasma membrane, has therefore been of prime interest for pharmaceutical development. A critical question in the activation of EPOR is whether differences in the relative arrangement of the soluble extracellular domains within the sEPOR dimers observed in the available crystal structures are truly indicative of active or inactive state as has been widely assumed 3 , 4 , 5 , 6 , 7 , When the crystal structures of the apo and EMP1-bound forms of the sEPOR domain were solved initially, it was proposed that distances between the termini observed crystallographically were distances that occurred on the cell surface.

In this model, ligand binding would separate the D1 domains and bring the D2 domains close together Fig. However, this model was proposed without any discussion of how the transmembrane domains might restrain the extracellular domains when embedded in a membrane.

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Do All X-ray Structures of Protein-Ligand Complexes Represent Functional States? EPOR, a Case Study

Not only has it been claimed that the precise distance between the terminal regions is crucial to the activity state of the sEPOR dimers, but also the angle formed between the receptor chains 3 , 4 , 5 , 6. The difference in this angle was claimed to account for the different activity of the ligand despite the distance of the C-termini between the sEPOR chains being practically identical.

This would suggest that not only are the individual domains rigid but that the binding of either EPO, EMP1, EMP33, or indeed any of the known agonists or antagonists, would need to hold the two receptor chains in a very precise relative orientation and to generate sufficient mechanical force to separate or bring together the transmembrane helices and the two associated JAK2 domains that consist of over amino acids each. This indicates that the range of motion seen within the individual receptor molecules in the simulations compares favorably with the variations observed crystallographically.

The range of motion observed in the simulations is also consistent with experimental studies that showed that the secondary structure of the receptor molecules in the EPOR dimer was not changed upon EPO binding using circular dichroism The aim was to determine whether the differences considered characteristic of activity based on the crystal structures were maintained in solution.

In general, the D2 domains moved close together in the simulations, except in three cases one agonist bound, one apo bound, and one antagonist bound where these domains separated Figs. As the EPO-binding site is occluded in the apo sEPOR dimer crystal structure, two further apo systems derived from EPO-bound conformations were investigated to determine whether the structure of the apo dimer as observed in the crystal structure could spontaneously form in solution.

In fact, the states sampled in the simulations were not obviously dependent on the nature or the presence of the ligand. They were, however, heavily influenced by the initial coordinates. This suggests that for these complexes the energy surface is relatively flat. Certainly, it was not possible to determine any structural differences that would support any of the potential models of receptor activation. This suggests the membrane, transmembrane domains, and intracellular regions all potentially play essential roles. Whether any of the agonists or antagonists examined would give rise to a distinct population of EPOR conformations given enough time, is unclear.

What is apparent is the ligands do not constrain the complexes to the unique conformations implied by the crystal structures. Thus, the available crystal structures are not an appropriate basis on which to propose a detailed mechanism whereby the ligands shift the conformation of the EPOR dimers on the cell surface between well-defined active and inactive states.

In both cases, D 2 d gave almost identical values to the respective initial values. The RMSD values for these systems calculated over the entire crystal lattice were about 0. The values plotted are from the final 2. Purple , green , red , aqua , magenta , orange , blue , crimson The asymmetric units, respectively. Black circle Initial values in the x-ray crystal structure; gray square values obtained from the structure of the average asymmetric unit.

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The aim of this study is not to cast aspersion on any specific structure but simply to highlight the difficulty and pitfalls when attempting to infer how a receptor or other protein may operate when only considering the structure of a protein within a crystal lattice. This is of particular importance in the case of membrane-associated proteins such as the EPOR and other cytokine receptors, where only the soluble extracellular domain is considered. In short, the differences observed in the crystals are unlikely to be physiologically significant.

Clearly, in the case of the EPOR, the influence of the transmembrane domains and the membrane environment must be considered when attempting to propose a mechanism of activation. Recently, a series of crystals was published suggesting alternative arrangements of the sEPOR dimer bound to diabodies with a range of proliferative activity 7. Such large distances are in stark contrast to the values seen with the native ligand EPO and the small EMPs, and would imply a mechanism of action very different from that proposed for closely related systems such as the growth hormone receptor.

In light of the simulations presented here, the physiological significance of the arrangement observed in these complexes should be treated with due caution. National Center for Biotechnology Information , U. Journal List Biophys J v. Published online Feb Corbett , 1 Alan E. Received Oct 14; Accepted Dec This article has been cited by other articles in PMC.

Energetics of Biological Macromolecules, Part C

Abstract Based on differences between the x-ray crystal structures of ligand-bound and unbound forms, the activation of the erythropoietin receptor EPOR was initially proposed to involve a cross-action scissorlike motion. Introduction The erythropoietin receptor EPOR is a cell-surface protein receptor and a member of the type-I cytokine receptor family. Open in a separate window. Table 2 Overview of the Systems Simulated. Receptor complex in a crystal lattice All simulations of the receptor complex in a crystal lattice were performed using the GROMACS simulation package 4.

Analysis RMSD The RMSD of the coordinates of the backbone with respect to a reference structure was calculated after performing a least-squares fit of the backbone atoms to the reference structure. Discussion The EPOR is a primary regulator of erythropoiesis but has also been recently identified in neurons where it helps protect against brain injury Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Crystals of the complex between human growth hormone and the extracellular domain of its receptor.

Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Functional mimicry of a protein hormone by a peptide agonist: An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation. Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Tuning cytokine receptor signaling by re-orienting dimer geometry with surrogate ligands. Structural insight into the transmembrane domain and the juxtamembrane region of the erythropoietin receptor in micelles.

Solution structure of the transmembrane domain of the mouse erythropoietin receptor in detergent micelles. Erythropoietin receptor activation by a ligand-induced conformation change.

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Revisiting the scissor-like mechanism of activation for the erythropoietin receptor. An automated force field topology builder ATB and repository: Molecular dynamics with coupling to an external bath. Interaction models for water in relation to protein hydration. Reidel; Dordrecht, The Netherlands: Only on your first 2 cashless orders on Amazon.

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Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology) Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)
Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology) Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)
Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology) Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)
Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology) Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)
Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology) Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)
Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology) Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)
Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology) Energetics of Biological Macromolecules, Part C: 323 (Methods in Enzymology)

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