Cooperative effects of potassium, magnesium, and magnesium-ADP on the release of Escherichia coli dihydrofolate reductase from the chaperonin GroEL

Previous investigation has shown that at 22 degrees C and in the presence o f the chaperonin GroEL, the slowest step in the refolding of Escherichia co li dihydrofolate reductase (EcDHFR) reflects release of a late folding inte rmediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 26 8.512-525). In this paper, we investigate the effects of potassium, magnesi um, and MgADP on the release of the EcDHFR late folding intermediate from G roEL. The data demonstrate that GroEL consists of at least two conformation al states, with apparent rate constants for EcDHFR release that differ by f our- to fivefold. In the absence of potassium, magnesium, and ADP, similar to 80-90% of GroEL resides in the form with the faster rate of release. Mag nesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-i nduced transition is observed only in the presence of potassium. MgADP at l ow concentrations (0-50 mu M) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium d ependent. Nearly identical results were obtained with a GroEL mutant that f orms only a single ring, demonstrating that these effects occur within a si ngle toroid of GroEL. In the presence of saturating magnesium, potassium, a nd MgADP, the apparent rate constant for the release of EcDHFR from wild-ty pe GroEL at 22 degrees C reaches a limiting value of 0.014 s(-1). For the s ingle ring mutant of GroEL, the rate of EcDHFR release under the same condi tions reaches a limiting value of 0.024 s(-1), suggesting that inter-ring n egative cooperativity exists for MgADP-induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a co nformational change leading to more rapid release of substrate protein.