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Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Keune WJ, Hausmann J, Bolier R, Tolenaars D, Kremer A, Heidebrecht T, Joosten RP, Sunkara M, Morris AJ, Matas-Rico E, Moolenaar WH, Oude Elferink RP, Perrakis A - Nat Commun (2016)

Bottom Line: Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA).ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function.Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

No MeSH data available.


Related in: MedlinePlus

TUDCA acts as a non-competitive inhibitor of LPC hydrolysis.(a) ATX lysoPLD activity with no inhibitor (black line and symbols) and with three concentrations (conc) of TUDCA (orange lines and symbols) as function of LPC(18:1) substrate conc. Modelling of all data (see the Methods for details) indicate that TUDCA acts as a partial noncompetitive inhibitor, with a Ki of 9±3 μM and residual activity of ∼40% towards LPC. (b) A cartoon of the ATX structure with bound TUDCA (orange carbons) in the tunnel and LPA (blue carbons) in the pocket, both shown as space filling models. (c,d) A zoom-in view showing the molecular surface of ATX at the TUDCA- and LPA-binding sites empty (c) and with bound TUDCA and LPA (d) as space filling models. (e) A zoom-in to a view along the tunnel axis, showing the characteristic L-shaped bile acid ring system and the bound LPA(18:1); the taurine tail has moved away from the active site to make space for the LPA; the active site zincs are visible to the right as grey spheres.
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f5: TUDCA acts as a non-competitive inhibitor of LPC hydrolysis.(a) ATX lysoPLD activity with no inhibitor (black line and symbols) and with three concentrations (conc) of TUDCA (orange lines and symbols) as function of LPC(18:1) substrate conc. Modelling of all data (see the Methods for details) indicate that TUDCA acts as a partial noncompetitive inhibitor, with a Ki of 9±3 μM and residual activity of ∼40% towards LPC. (b) A cartoon of the ATX structure with bound TUDCA (orange carbons) in the tunnel and LPA (blue carbons) in the pocket, both shown as space filling models. (c,d) A zoom-in view showing the molecular surface of ATX at the TUDCA- and LPA-binding sites empty (c) and with bound TUDCA and LPA (d) as space filling models. (e) A zoom-in to a view along the tunnel axis, showing the characteristic L-shaped bile acid ring system and the bound LPA(18:1); the taurine tail has moved away from the active site to make space for the LPA; the active site zincs are visible to the right as grey spheres.

Mentions: Testing the inhibitory effect of different TUDCA concentrations against increasing concentrations of LPC, showed a reduction in Vmax with no significant change in the kM of the reaction (Fig. 5a); consistently, kinetic modelling confirmed that a competitive model has a probability of <0.01% to be correct (see the Methods for details and equations). As TUDCA is a partial inhibitor of ATX (Fig. 3b), we used a partial mixed inhibition model to explain the kinetic data (see the Methods for details). Kinetic modelling suggested that while TUDCA has an appreciable ϰI of 9±3 μM, even at saturating concentrations ∼40% of ATX activity towards LPC is retained. Crucially, mathematical modelling indicated that TUDCA is a noncompetitive inhibitor, implying that LPC or LPA can co-exist in a ternary complex with ATX.


Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Keune WJ, Hausmann J, Bolier R, Tolenaars D, Kremer A, Heidebrecht T, Joosten RP, Sunkara M, Morris AJ, Matas-Rico E, Moolenaar WH, Oude Elferink RP, Perrakis A - Nat Commun (2016)

TUDCA acts as a non-competitive inhibitor of LPC hydrolysis.(a) ATX lysoPLD activity with no inhibitor (black line and symbols) and with three concentrations (conc) of TUDCA (orange lines and symbols) as function of LPC(18:1) substrate conc. Modelling of all data (see the Methods for details) indicate that TUDCA acts as a partial noncompetitive inhibitor, with a Ki of 9±3 μM and residual activity of ∼40% towards LPC. (b) A cartoon of the ATX structure with bound TUDCA (orange carbons) in the tunnel and LPA (blue carbons) in the pocket, both shown as space filling models. (c,d) A zoom-in view showing the molecular surface of ATX at the TUDCA- and LPA-binding sites empty (c) and with bound TUDCA and LPA (d) as space filling models. (e) A zoom-in to a view along the tunnel axis, showing the characteristic L-shaped bile acid ring system and the bound LPA(18:1); the taurine tail has moved away from the active site to make space for the LPA; the active site zincs are visible to the right as grey spheres.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834639&req=5

f5: TUDCA acts as a non-competitive inhibitor of LPC hydrolysis.(a) ATX lysoPLD activity with no inhibitor (black line and symbols) and with three concentrations (conc) of TUDCA (orange lines and symbols) as function of LPC(18:1) substrate conc. Modelling of all data (see the Methods for details) indicate that TUDCA acts as a partial noncompetitive inhibitor, with a Ki of 9±3 μM and residual activity of ∼40% towards LPC. (b) A cartoon of the ATX structure with bound TUDCA (orange carbons) in the tunnel and LPA (blue carbons) in the pocket, both shown as space filling models. (c,d) A zoom-in view showing the molecular surface of ATX at the TUDCA- and LPA-binding sites empty (c) and with bound TUDCA and LPA (d) as space filling models. (e) A zoom-in to a view along the tunnel axis, showing the characteristic L-shaped bile acid ring system and the bound LPA(18:1); the taurine tail has moved away from the active site to make space for the LPA; the active site zincs are visible to the right as grey spheres.
Mentions: Testing the inhibitory effect of different TUDCA concentrations against increasing concentrations of LPC, showed a reduction in Vmax with no significant change in the kM of the reaction (Fig. 5a); consistently, kinetic modelling confirmed that a competitive model has a probability of <0.01% to be correct (see the Methods for details and equations). As TUDCA is a partial inhibitor of ATX (Fig. 3b), we used a partial mixed inhibition model to explain the kinetic data (see the Methods for details). Kinetic modelling suggested that while TUDCA has an appreciable ϰI of 9±3 μM, even at saturating concentrations ∼40% of ATX activity towards LPC is retained. Crucially, mathematical modelling indicated that TUDCA is a noncompetitive inhibitor, implying that LPC or LPA can co-exist in a ternary complex with ATX.

Bottom Line: Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA).ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function.Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

No MeSH data available.


Related in: MedlinePlus