Probing Open Conformation of GroEL Rings by Cross-linking Reveals Single and Double Open Ring Structures of GroEL in ADP and ATP

Two heptamer rings of chaperonin GroEL undergo opening-closing conformational transition in the reaction cycle with the aid of GroES and ATP. We introduced Cys into the GroEL subunit at Ala-384 and Ser-509, which are very close between adjacent GroEL subunits in the open heptamer ring but far apart in the closed heptamer ring. The open ring-specific inter-subunit cross-linking between these Cys indicated that the number of rings in open conformation in GroEL was two in ATP (Gro-ELOO), one in ADP (GroEL(O)), and none in the absence of nucleotide. ADP showed an inhibitory effect on ATP-induced generation of GroEL(OO). The isolated GroEL(O) and GroEL(OO), which lost any bound nucleotide, could bind GroES to form a bullet-shaped 1:1 GroEL-GroES complex and a football-shaped 1: 2 GroEL-GroES complex, respectively, even without the addition of any nucleotide. Substrate protein was unable to form a stable complex with GroEL(OO) and did not stimulate ATPase activity of GroEL. These results favor a model of the GroEL reaction cycle that includes a football complex as a critical intermediate.