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Conformational thermostabilisation of corticotropin releasing factor receptor 1.

Kean J, Bortolato A, Hollenstein K, Marshall FH, Jazayeri A - Sci Rep (2015)

Bottom Line: Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation.Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents.Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation.

View Article: PubMed Central - PubMed

Affiliation: Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.

ABSTRACT
Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation.

No MeSH data available.


CRF1R WT and StaR molecular dynamics analysis.CRF1R structural superimposition of the starting and final conformation (after 100 ns explicit all-atom MD in an OG-water-micelle environment) for the StaR (panel (A)) and WT (panel (B)). Only the helical bundle backbone is shown as ribbon together with the ligand CP-376395. The initial conformation is coloured in green and magenta respectively for the StaR and the WT, while the final state is respectively in cyan and yellow. (panel (C)) Molecular destabilizing effects at the end of the CRF1R WT MD simulation (in yellow) of the residues I2775.44, Y3096.35, F3306.56 and Y3637.57 (all mutated to Ala in the StaR receptor). For clarity only TM5, TM6 and TM7 are shown. The backbone of the starting conformation is included in magenta as ribbon. I2775.44, F3306.56, T3266.52, L3517.45 and the ligand CP-376395 are shown as space filling, while Y3096.35, Y3637.57, P3216.47, S3497.43 and 2 OG molecules in stick representation.
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f4: CRF1R WT and StaR molecular dynamics analysis.CRF1R structural superimposition of the starting and final conformation (after 100 ns explicit all-atom MD in an OG-water-micelle environment) for the StaR (panel (A)) and WT (panel (B)). Only the helical bundle backbone is shown as ribbon together with the ligand CP-376395. The initial conformation is coloured in green and magenta respectively for the StaR and the WT, while the final state is respectively in cyan and yellow. (panel (C)) Molecular destabilizing effects at the end of the CRF1R WT MD simulation (in yellow) of the residues I2775.44, Y3096.35, F3306.56 and Y3637.57 (all mutated to Ala in the StaR receptor). For clarity only TM5, TM6 and TM7 are shown. The backbone of the starting conformation is included in magenta as ribbon. I2775.44, F3306.56, T3266.52, L3517.45 and the ligand CP-376395 are shown as space filling, while Y3096.35, Y3637.57, P3216.47, S3497.43 and 2 OG molecules in stick representation.

Mentions: To better understand the difference in thermostability between the CRF1R StaR and the WT receptor we analysed their conformational changes occurring after 100 ns MD at 10 °C in the harsh detergent OG (Sup. Figure 4, E). In these conditions CRF1R StaR is stable, whilst the WT receptor quickly unfolds (Table 1). In both systems the detergent molecules create a stable micelle around the hydrophobic regions of the TM domain. However, the helical bundle shows a difference in macroscopic behaviour between the StaR and the WT receptor (Figs 4A,B). In the first case, the crystallographic conformation is very stable, while the three independent WT simulations show the initial signs of instability and unfolding even in this relative short time frame. In this system the initial steps of the receptor unfolding are variable, but generally involve TM4, TM5, TM6 and TM7 (Table 4). The increase in the TM stability of the StaR compared to that of WT during the simulations appeared variable and not linked to the number of mutations in the helices. This supports the conclusion that the effect of the StaR mutations on the conformational rigidity is complex, and relates to the whole helical bundle, not just a single TM in isolation. Particularly remarkable was the difference in TM5, TM6 and TM7 conformational stability between WT and the StaR. During the WT MD simulations the presence of isoleucine at position 2775.44 (mutated to Ala in the StaR receptor) can promote the insertion of OG molecules between TM5 and TM6 close to the CP-376395 binding site (Figs 4C). In that region, TM6 shows a distorted conformation close to P3216.47 as a result of opposing forces acting at the extracellular and intracellular sides, in addition to the OG insertion. In the extracellular portion of the receptor we identified the hydrophobic collapse of F3306.56 on T3266.52 and L3517.45. The intracellular conformation of TM6 and TM7 are kept close to the helical bundle by interactions created by Y3096.35 and Y3637.57 with TM2 and TM3. Together these interactions cause a conformational strain, resulting in the bending of TM6 at position 6.47, followed by the insertion of the OG molecule. This instability is not detected in the StaR MD simulation probably as a consequence of the StaR mutations I277A5.44, Y309A6.35 and Y363A7.57.


Conformational thermostabilisation of corticotropin releasing factor receptor 1.

Kean J, Bortolato A, Hollenstein K, Marshall FH, Jazayeri A - Sci Rep (2015)

CRF1R WT and StaR molecular dynamics analysis.CRF1R structural superimposition of the starting and final conformation (after 100 ns explicit all-atom MD in an OG-water-micelle environment) for the StaR (panel (A)) and WT (panel (B)). Only the helical bundle backbone is shown as ribbon together with the ligand CP-376395. The initial conformation is coloured in green and magenta respectively for the StaR and the WT, while the final state is respectively in cyan and yellow. (panel (C)) Molecular destabilizing effects at the end of the CRF1R WT MD simulation (in yellow) of the residues I2775.44, Y3096.35, F3306.56 and Y3637.57 (all mutated to Ala in the StaR receptor). For clarity only TM5, TM6 and TM7 are shown. The backbone of the starting conformation is included in magenta as ribbon. I2775.44, F3306.56, T3266.52, L3517.45 and the ligand CP-376395 are shown as space filling, while Y3096.35, Y3637.57, P3216.47, S3497.43 and 2 OG molecules in stick representation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4498186&req=5

f4: CRF1R WT and StaR molecular dynamics analysis.CRF1R structural superimposition of the starting and final conformation (after 100 ns explicit all-atom MD in an OG-water-micelle environment) for the StaR (panel (A)) and WT (panel (B)). Only the helical bundle backbone is shown as ribbon together with the ligand CP-376395. The initial conformation is coloured in green and magenta respectively for the StaR and the WT, while the final state is respectively in cyan and yellow. (panel (C)) Molecular destabilizing effects at the end of the CRF1R WT MD simulation (in yellow) of the residues I2775.44, Y3096.35, F3306.56 and Y3637.57 (all mutated to Ala in the StaR receptor). For clarity only TM5, TM6 and TM7 are shown. The backbone of the starting conformation is included in magenta as ribbon. I2775.44, F3306.56, T3266.52, L3517.45 and the ligand CP-376395 are shown as space filling, while Y3096.35, Y3637.57, P3216.47, S3497.43 and 2 OG molecules in stick representation.
Mentions: To better understand the difference in thermostability between the CRF1R StaR and the WT receptor we analysed their conformational changes occurring after 100 ns MD at 10 °C in the harsh detergent OG (Sup. Figure 4, E). In these conditions CRF1R StaR is stable, whilst the WT receptor quickly unfolds (Table 1). In both systems the detergent molecules create a stable micelle around the hydrophobic regions of the TM domain. However, the helical bundle shows a difference in macroscopic behaviour between the StaR and the WT receptor (Figs 4A,B). In the first case, the crystallographic conformation is very stable, while the three independent WT simulations show the initial signs of instability and unfolding even in this relative short time frame. In this system the initial steps of the receptor unfolding are variable, but generally involve TM4, TM5, TM6 and TM7 (Table 4). The increase in the TM stability of the StaR compared to that of WT during the simulations appeared variable and not linked to the number of mutations in the helices. This supports the conclusion that the effect of the StaR mutations on the conformational rigidity is complex, and relates to the whole helical bundle, not just a single TM in isolation. Particularly remarkable was the difference in TM5, TM6 and TM7 conformational stability between WT and the StaR. During the WT MD simulations the presence of isoleucine at position 2775.44 (mutated to Ala in the StaR receptor) can promote the insertion of OG molecules between TM5 and TM6 close to the CP-376395 binding site (Figs 4C). In that region, TM6 shows a distorted conformation close to P3216.47 as a result of opposing forces acting at the extracellular and intracellular sides, in addition to the OG insertion. In the extracellular portion of the receptor we identified the hydrophobic collapse of F3306.56 on T3266.52 and L3517.45. The intracellular conformation of TM6 and TM7 are kept close to the helical bundle by interactions created by Y3096.35 and Y3637.57 with TM2 and TM3. Together these interactions cause a conformational strain, resulting in the bending of TM6 at position 6.47, followed by the insertion of the OG molecule. This instability is not detected in the StaR MD simulation probably as a consequence of the StaR mutations I277A5.44, Y309A6.35 and Y363A7.57.

Bottom Line: Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation.Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents.Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation.

View Article: PubMed Central - PubMed

Affiliation: Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.

ABSTRACT
Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation.

No MeSH data available.