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  • br Structural Homology and Functional Implications Amino aci

    2021-09-09


    Structural Homology and Functional Implications Amino PD 0332991 sequence analyses of Disp and the Shh receptor Ptch indicate that both proteins share structural homology with a family of bacterial efflux pumps that function in resistance, nodulation, and division (RND) transporter complexes (Figure 1A) 12., 13.. In bacteria, these antiporters work as trimers and use the proton motive force to move a range of small-molecule substrates across membranes [20]. A characteristic of such proteins is the topology of their 12 transmembrane (TM) domains, which are arranged into two pseudosymmetrical halves each containing six TM segments and one large extracellular globular domain (Figure 1B) 20., 21.. Although the Disp structure has not yet been solved, recent cryo-EM studies revealed that Ptch possesses stereotypical RND membrane topology and closely resembles structures of the bacterial aerobic respiration control sensor protein (ArcB) transporter and the vertebrate cholesterol transporter Niemann–Pick type C1 (NPC1) (Figure 1C) 22., 23., 24., 25.. Disp, Ptch, and NPC1 all contain sterol-sensing domains (SSDs), which are unique to proteins that bind, transport, or respond to cellular sterols [26]. In both Ptch and Disp, the SSD spans TM2–TM6 and contains a conserved GxxDD motif in TM4 (Figure 1B). In bacterial RND efflux pumps, similar motifs work with a second motif in TM10 to coordinate protons and move substrate 20., 27.. A second conserved GxxxD motif is present in Disp TM10, and when disrupted along with the TM4 motif prevented the release of Hh from ligand-producing cells. Thus, it was initially hypothesized that Disp would use a TM4/TM10 proton-binding mechanism similar to RND transporters to release Hh ligands [13]. However, more recent work demonstrated that while mutation of the TM4 motif was sufficient to impair Hh release, mutation of the TM10 GxxxD on its own was not [28]. Thus, compromised function of the TM4/TM10 compound mutant may be the result of disrupted SSD function rather than the blocking of a TM4/TM10-mediated proton-driven transport activity. Despite their predicted structural homology, sequence homology between Disp, Ptch, and NPC1 is limited predominantly to their SSDs [26]. The recent Ptch structures support that, similar to NPC1, its SSD binds and potentially transports a sterol molecule (Figure 1C) 22., 23., 24., 25.. Structural examination of Shh in complex with Ptch revealed the primary interface between Shh and Ptch to occur by the amino-terminal fatty acid modification of the ligand inserting into a channel formed between the two large extracellular domains (ECDs) of Ptch [22]. SSD mutations that block sterol binding to Ptch shifted the conformation of the ECDs to compromise Shh association, potentially indicating that sterol loading into the SSD may impact fatty acid access to the ECD channel 22., 24.. The role of the Disp SSD in Shh release is not yet clear. However, a recent biochemical interrogation suggested that the SSD may facilitate Disp–Shh binding by directly associating with the carboxyl-terminal cholesterol modification. To release ligand, Disp was hypothesized to transfer the sterol tag to the secreted glycoprotein signal peptide, CUB, and epidermal growth factor-like domain-containing protein 2 (Scube2), a vertebrate-specific Disp cofactor that significantly enhances Shh membrane extraction [29]. This model is supported by the ability of both Disp and Scube2 to individually coimmunoprecipitate with cholesterol-modified Shh but not with a truncated ShhN molecule that lacks the carboxyl-terminal sterol. Further, Shh modified with photoactivatable cholesterol coimmunoprecipitated with Disp and Scube2 under denaturing conditions post-crosslinking, consistent with Shh being attached to each of the deployment proteins by sterol [29]. Thus, Disp was proposed to transfer cholesterol-modified Shh to Scube2 in a manner analogous to how NPC1 transfers cholesterol to NPC2 29., 30.. Although this is a logical hypothesis, results from a second study of Disp and Scube2 indicated that the amino-terminal fatty acid modification on Shh may be the Scube2-associated lipid [31]. Shh binding to Scube2 was not strictly dependent on fatty acylation, but biochemical analysis of their association revealed that palmitoylated Shh bound Scube2 more tightly than a mutant lacking the palmitate moiety. Additionally, unmodified Shh was released from membranes by Disp and Scube2 more slowly than fatty-acylated ligand, supporting a direct contribution of palmitate to Scube2-assisted membrane extraction [31]. These results suggest an alternative model in which the Disp SSD could bind the cholesterol modification, allowing Scube2 to collect Shh from Disp by binding the amino-terminal fatty acid. Given the recent discovery that free cholesterol binding to the Ptch SSD promotes Shh binding 22., 24., it should also be considered that free cholesterol could bind the Disp SSD to similarly influence its ability to bind Hhs or transfer them to Scube2.