Figure 4. ASMase^sap–ASMase^cat interactions.

(a) Superposition of the two molecules in the asymmetric unit of the closed form. The reference portion for the superposition is the catalytic domain (light green surface). The saposin domains (worm representation) are shifted by roughly 8 Å, and the linker connecting the saposin domain to the catalytic domain is disordered in one of the molecules (blue). (b) Close-up of the interface formed between open ASMase^sap (pink) and ASMase^cat (green). Inset: yellow shading marks the surface buried on interface formation. Active site loops in contact with the interface are coloured black. Interface mutations tested are marked by an asterisk. (c) Activity at pH 5 of wild type (WT) and mutants on liposomes containing sphingomyelin (SM). Bar colours correspond to ASMase^cat (green) or ASMase^sap (pink) mutants. Activity is normalized to WT enzyme. 100% activity=537 μM SM hydrolyzed per μM protein per h on anionic liposomes. Data are the means and s.d.'s of two independent experiments performed in triplicates. (d) Activity at pH 5 of WT and mutants on the small-molecule substrate bNPP. Activity is normalized to the WT enzyme. 100% activity=1.76 μM bNPP hydrolyzed per nM protein per h. Data are from two to five experiments performed in triplicates. (e) Effect of detergents on activity against bNPP. Data are from two independent experiments performed in triplicates. OGP, octyl-β-D-glucopyranoside; NP-40, Tergitol-type NP-40; TX-100, Triton X-100. For the latter two detergents, 0.1 and 1 mM represent concentrations below and above their critical micellar concentrations.