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Making structural sense of dimerization interfaces of delta opioid receptor homodimers.

Johnston JM, Aburi M, Provasi D, Bortolato A, Urizar E, Lambert NA, Javitch JA, Filizola M - Biochemistry (2011)

Bottom Line: Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities.Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer.Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York 10029, United States.

ABSTRACT
Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed "4" dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

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Free energy of 4 (red) or 4/5 (blue) interprotomeric arrangements of DOR restrained in their relative orientation by square-well potentials applied on the rotational angles. For the sake of simplicity, the curves are shifted to assign 0 values to monomeric states (r ≥ 4.5 nm).
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fig5: Free energy of 4 (red) or 4/5 (blue) interprotomeric arrangements of DOR restrained in their relative orientation by square-well potentials applied on the rotational angles. For the sake of simplicity, the curves are shifted to assign 0 values to monomeric states (r ≥ 4.5 nm).

Mentions: To estimate the relative stability of the 4/5 dimer versus the 4 dimer, we performed umbrella sampling simulations of the CG 4/5 dimer in an explicit CG POPC/10% cholesterol environment and compared the results of these simulations with analogous simulations we recently conducted with the 4 dimer (17). As shown in Figure 5, a single minimum was identified for the 4/5 dimer (blue line) at an energy higher than those of the two energetically and structurally similar dimeric minima previously identified for the 4 dimer (red line) (17). Using the calculated free energy area for the 4/5 dimer from Figure 5 and the formalism originally described by Roux and co-workers (47−49) and recently applied by us to the 4 dimer of DOR (see details in ref (17)), we estimated a dimerization constant (KDimer) of ≅0.05 μm2 for the identified lowest-energy 4/5 dimer compared to the recently reported value of 1.02 μm2 for the 4 dimer (17). Using this dimerization constant in combination with a diffusion coefficient of 0.08 μm2/s determined experimentally for MOR (15), we calculated a half-time of 0.2 s for the 4/5 dimer, compared to the value of 4.4 s previously reported for the 4 dimer (17).


Making structural sense of dimerization interfaces of delta opioid receptor homodimers.

Johnston JM, Aburi M, Provasi D, Bortolato A, Urizar E, Lambert NA, Javitch JA, Filizola M - Biochemistry (2011)

Free energy of 4 (red) or 4/5 (blue) interprotomeric arrangements of DOR restrained in their relative orientation by square-well potentials applied on the rotational angles. For the sake of simplicity, the curves are shifted to assign 0 values to monomeric states (r ≥ 4.5 nm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050604&req=5

fig5: Free energy of 4 (red) or 4/5 (blue) interprotomeric arrangements of DOR restrained in their relative orientation by square-well potentials applied on the rotational angles. For the sake of simplicity, the curves are shifted to assign 0 values to monomeric states (r ≥ 4.5 nm).
Mentions: To estimate the relative stability of the 4/5 dimer versus the 4 dimer, we performed umbrella sampling simulations of the CG 4/5 dimer in an explicit CG POPC/10% cholesterol environment and compared the results of these simulations with analogous simulations we recently conducted with the 4 dimer (17). As shown in Figure 5, a single minimum was identified for the 4/5 dimer (blue line) at an energy higher than those of the two energetically and structurally similar dimeric minima previously identified for the 4 dimer (red line) (17). Using the calculated free energy area for the 4/5 dimer from Figure 5 and the formalism originally described by Roux and co-workers (47−49) and recently applied by us to the 4 dimer of DOR (see details in ref (17)), we estimated a dimerization constant (KDimer) of ≅0.05 μm2 for the identified lowest-energy 4/5 dimer compared to the recently reported value of 1.02 μm2 for the 4 dimer (17). Using this dimerization constant in combination with a diffusion coefficient of 0.08 μm2/s determined experimentally for MOR (15), we calculated a half-time of 0.2 s for the 4/5 dimer, compared to the value of 4.4 s previously reported for the 4 dimer (17).

Bottom Line: Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities.Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer.Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York 10029, United States.

ABSTRACT
Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed "4" dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

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