Fig. 5. Structural analysis and functional validation of the Prc^SK-NlpI-mMepS complex.

A Two orthogonal views of the Prc^SK-NlpI-mMepS complex are depicted, highlighting the interaction site between Prc^SK and NlpI in cartoon representation. 2F_o - F_c maps of TPR2b and h1 from Prc^SK are contoured at 1.0 σ as grey and pale green mesh, with the first and last residues labeled (TPR2b: F112 and D126, h1: T39 and S55). NlpI is represented in gray, Prc^SK in yellow and pale green, mMepS1 in salmon and magenta and mMepS2 in cyan and golden. The Prc^SK protease forms a bowl-like structure with a lid-shaped PDZ domain connected through a substrate-recognizing hinge. Owing to the flexibility of PDZ domains, cartoon models of PDZ domain are shown to emphasize the high dynamics of this region. The missing PDZ-connected hinge is depicted as a red dotted curve, and the dotted double arrow indicates the motion of PDZ domain. B A size-exclusion chromatogram of the Prc^SK-NlpI-mMepS complex is presented, featuring the radius of gyration (Rg) as a grey rectangle and zero-angle scattering intensity (I₀) as a purple dotted line, plotted along with the absorbance at 280 nm (I_UV) represented by a green line. The elution time is calculated by multiplying the number of SAXS frames by the frame interval of 2.1 seconds. The exposure time is set at 2 seconds per frame, with a wait time of 0.1 seconds per frame interval. C In vitro degradation assays of mMepS and mutant proteins by the Prc-NlpI proteolytic system were conducted. Purified MepS proteins were incubated with Prc in the company of NlpI at 37 °C. Protein samples were collected at the indicated time points and analyzed using SDS-PAGE gels followed by Coomassie blue staining. The residual levels of substrate were determined by quantifying the protein band intensity using ImageJ. The data represent a single experiment out of three independent (n = 3) measurements. Source data are provided as a Source Data file.