Figure 5. A₂ binds MurA in a substrate-dependent manner.

(A) MBP-A₂ associates with MurA in a closed conformational state. Eluates from amylose magnetic bead fractionation experiments were Western blotted and probed with the α-MurA antibody. MBP-A₂ was incubated with MurA under various substrate conditions: No substrate (−), PEP (P), UDP-NAG (U), and both substrates (U/P). MurA^L138Q rat mutant was tested in parallel. (B) Unbound fractions of panel A experiments. (C) Fusion cleavage analysis of A₂-MurA binding. MBP-A₂ was cleaved with TEV protease in the absence or presence of MurA under various substrate conditions (same as in panel A). Binding was assessed as A₂ solubility after centrifugation: Total fraction (T), supernatant after centrifugation (S), and pellet fraction (P). Samples were resolved on SDS-PAGE. MBP-A₂ has an apparent MW of 100 kDa. Cleaved A₂ runs at 50 kDa with the MBP running at the same apparent MW as MurA (~52 kDa). (D) A₂ binds MurA liganded to UDP-NAG. Western blot analysis of fusion cleavage assays. Blots were probed with the α-A₂ antibody. Binding was tested with substrate conditions as in panel C. (E) A₂ does not bind the tetrahedral intermediate state of MurA. UDP-NAG and Fosfomycin (U/F) liganded MurA and MurA^D305A binding and immunoblotting was also performed in parallel as in panel D.