Abstract
The recent mu-opioid receptor (MOPr) and kappa-opioid receptor (KOPr) crystal structures have inspired hypotheses of physiologically relevant dimerization contacts, specifically: a closely packed interface involving transmembrane (TM) helices TM5 and TM6, and a less compact interface, involving TM1, TM2, and helix 8 (H8). While the former was only found in MOPr crystals, similar arrangements of the latter were identified for both KOPr and MOPr. The relevance of these interfaces outside of a crystal lattice is called into question by the possibility that they might be influenced by the specific crystallization conditions. In this study, we have employed umbrella sampling molecular dynamics simulations of coarse-grained representations of the interacting MOPr or KOPr crystallographic structures, in the absence of the T4 lysozyme, and in an explicit lipid-water environment, to determine the strength of receptor dimerization at the different crystallographic interfaces. We note that the shape of the interface plays a dominant role in the strength of the interaction, and the pattern of contacting residues defines the shape of the potential of mean force. This information can be used to guide experiments aimed at exploring the role of dimerization in opioid receptor function.
Highlights
Following several breakthroughs in crystallization techniques for G Protein-Coupled Receptors (GPCRs), T4 lysozyme (T4L)-stabilized constructs and lipid cubic phase environments [1,2,3], several GPCR crystallographic structures have been solved in recent years, including those of opioid receptors [4,5,6,7]
We evaluated the strength of dimerization of mu-opioid receptor (MOPr) and kappa-opioid receptor (KOPr) interfaces involving TM1, TM2, and helix 8 (H8), as well as the MOPr interface involving TM5 and TM6
In the more compact TM5,6/TM5,6 crystallographic interface of MOPr, compared to that involving TM1, TM2, and H8, contacts are formed along the length of both of the TM5 and TM6 membrane spanning helices and the separation between the centers of mass (COMs) of the helix bundles is,3.3 nm
Summary
Following several breakthroughs in crystallization techniques for G Protein-Coupled Receptors (GPCRs), T4 lysozyme (T4L)-stabilized constructs and lipid cubic phase environments [1,2,3], several GPCR crystallographic structures have been solved in recent years, including those of opioid receptors [4,5,6,7]. A recent analysis [16] applying the Evolutionary Protein-Protein Interface Classifier to distinguish biological interfaces from some of the recently characterized crystallographic interfaces of family A GPCRs, including KOPr, concluded that, with the exception of the class F Smoothened receptor, none of the analyzed crystallographic interfaces of GPCRs exhibit the signature geometrical packing and evolution patterns expected for high-affinity, physiologically-relevant interfaces While this is interesting, the results of an evolutionary analysis are limited by the number of relatively close sequence homologs that are available for the alignment, the uniformity of the distribution of identities in the homologs, and the simplicity of a sequence-based approach, which takes into account neither the energetics underlying the dimerization process, nor the role of the environment. We sought to investigate both the viability and relative stability of the crystallographic interfaces identified for the MOPr and the KOPr in an explicit lipid-water environment, and in the absence of the T4L, in an effort to start prioritizing mutagenesis experiments aimed at exploring the role of dimerization in opioid receptor function
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