The GroE chaperonins assist substrate protein (SP) folding by cycling through several conformational states. With each cycle the SP is, in turn, captured, unfolded, briefly encapsulated (t1/2 ∼ 1 s), and released by the chaperonin complex. The protein-folding functional form is the US-football-shaped GroEL:GroES2 complex. We report structures of two such "football" complexes to ∼ 3.7-Å resolution; one is empty whereas the other contains encapsulated SP in both chambers. Although encapsulated SP is not visible on the electron density map, using calibrated FRET and order-of-addition experiments we show that owing to SP-catalyzed ADP/ATP exchange both chambers of the football complex encapsulate SP efficiently only if the binding of SP precedes that of ATP. The two rings of GroEL thus behave as a parallel processing machine, rather than functioning alternately. Compared with the bullet-shaped GroEL:GroES1 complex, the GroEL:GroES2 football complex differs conformationally at the GroEL-GroES interface and also at the interface between the two GroEL rings. We propose that the electrostatic interactions between the ε-NH(3+) of K105 of helix D in one ring with the negatively charged carboxyl oxygen of A109 at the carboxyl end of helix D of the other ring provide the structural basis for negative inter-ring cooperativity.
Keywords: crystal structure; encapsulation; order-of-ligand-addition; symmetric.