Biochemical Assays to Directly Assess Smoothened Activation by a Conformationally Sensitive Nanobody

Abstract

The Hedgehog signaling pathway coordinates early development and is important in various cancers. Classic approaches to test pathway activation rely on transcriptional readouts or ciliary accumulation of specific pathway components. Although these assays have laid the foundation for studying Hedgehog pathway activation, they integrate the complex molecular actions of the transporter Patched and the seven transmembrane protein Smoothened. Though it is clear that cellular sterols are critical for pathway activity, direct dissection of which sterols drive Smoothened activity is precluded by the complex biosynthetic pathways responsible for cellular sterols. Here we describe a direct method of measuring Smoothened activity in vitro. This assay measures the binding of Smoothened to NbSmo8, a single-domain antibody that is selective for the active-state of Smoothened. Binding of purified Smoothened, reconstituted with specific sterols, to fluorescently labeled NbSmo8 can be rapidly evaluated using a fluorescence-detection size-exclusion chromatography assay. This approach enables a reductionist approach to precisely interrogate the regulatory activities of cellular lipids and sterols during Hedgehog signaling.

Keywords: Fluorescence size-exclusion chromatography; Hedgehog signaling; Nanobody; NbSmo8; Smoothened activation.

Fig 1.

Schematic diagram of Fluorescent size-exclusion chromatography (FSEC) experiment. Smoothened (SMO; in blue) is mixed with NbSmo8 (in gold) labeled with Alexa647 (in red) and subjected to a size exclusion column connected to a fluorescence detector. Traces of 2 μM NbSmo8–Alexa647 incubated with 1 μM SMO in the presence of L-MNG/CHS micelles (grey) or L-MNG/cholesterol micelles (green). NbSmo8-Alexa647 co-elutes with SMO in the presence of cholesterol, but not CHS, with a retention volume of 12.6 mL.