Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling

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.

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

“…The most likely candidate for an LPA allosteric binding site is the ATX tunnel, which has been shown to bind natural steroids 16 and, importantly, has also been suggested to bind LPA in a series of crystal structures reported in 11 ; where the authors showed density for about six carbons of the acyl chain of LPA, bound to the tunnel. Aiming to confirm this interaction, we designed a series of biochemical assays studying the modulation of ATX enzymatic activity by LPA in the presence of different well-characterized inhibitors that bind in the orthosteric and allosteric sites.…”

“…The first of these inhibitors was the bile salt TUDCA, an inhibitor with IC 50 of 11 µM, which we previously reported to bind the allosteric site 16 . The effect of LPA activation under conditions where ATX is partially inhibited by three TUDCA concentrations was measured.…”

“…The discovery of the tunnel as an allosteric site which can modulate ATX activity 16 offered an opportunity to extend this model. This also made it possible to explain previous observations that we were unable to rationalize before, namely the LPA mediated ATX activation and the lag phase that we have consistently observed in our choline release assays for LPA hydrolysis.…”

“…Activity was measured by a coupled reaction with 1 U ml -1 choline oxidase and 2U ml 16 . Due to the presence of a lag phase during the first 10 min of ATX-dependent LPC hydrolysis (Fig.1B), the subsequent linear slope was used to perform all analyses.…”

“…The work on type III and type IV inhibitors has highlighted further the tunnel as a novel allosteric site to modulate ATX activity. This modulation has been hypothesized to occur by disturbing the catalytic cleavage of substrates and/or by modulating LPA product uptake and the delivery to its cognate LPA receptors 16 . In such a mechanism, ATX not only drives the formation of LPA but also ensures specificity in LPA signalling.…”