Role of Polyubiquitin Chain Flexibility in the Catalytic Mechanism of Cullin-RING Ubiquitin Ligases.

Abstract

Cullin-RING ubiquitin ligases are a diverse family of ubiquitin ligases that catalyze the synthesis of K48-linked polyubiquitin (polyUb) chains on a variety of substrates, ultimately leading to their degradation by the proteasome. The cullin-RING enzyme scaffold processively attaches a Ub molecule to the distal end of a growing chain up to lengths of eight Ub monomers. However, the molecular mechanism governing how chains of increasing size are built using a scaffold of largely fixed dimensions is not clear. We developed coarse-grained molecular dynamics simulations to describe the dependence of kcat for cullin-RING ligases on the length and flexibility of the K48-linked polyUb chain attached to the substrate protein, key factors that determine the rate of subsequent Ub attachment to the chain, and therefore, the ensuing biological outcomes of ubiquitination. The results suggest that a number of regulatory mechanisms may lead to variations in the rate of chain elongation for different cullin-RING ligases. Specifically, modulation of the distance between the target lysine and the phosphodegron sequence of the substrate, the distance between the substrate lysine and the active site cysteine of the Ub conjugation enzyme (E2) bound to the cullin-RING scaffold, and flexibility of the bound E2 can lead to significant differences in the processing of K48-linked chains on substrates, potentially leading to differences in biological outcomes.