SCRIB inhibits PP1 phosphatase activity without blocking or structurally altering its active site.

Protein phosphorylation is a fundamental mechanism regulating cellular function, governed by the opposing actions of kinases and phosphatases. The substrate specificity and catalytic activity of protein phosphatase 1 (PP1), a major serine/threonine phosphatase, results from its interactions with more than 200 regulatory proteins. However, the mechanisms employed remain largely unknown. Here, we present the 3.5 Å cryo-electron microscopy structure of the complex between PP1 and the tumor suppressor and negative ERK-signaling regulator scribble homologue (SCRIB), revealing an unprecedented inhibitory mechanism. SCRIB engages PP1 via a multipronged interface composed of its leucine-rich repeat (LRR) domain, its first PDZ domain, and its flanking regions, where the 705KGVSF sequence acts as a non-canonical PP1-binding RVxF motif. Enzymatic kinetics demonstrate that SCRIB potently dampens catalytic turnover (kcat) while preserving PP1 in a fully ion-loaded, "active-like" state. Structural, computational, and evolutionary analyses suggest that SCRIB achieves this inhibition by allosterically modulating the electrostatic landscape and rigidifying the dynamic properties of the PP1 core. These findings identify SCRIB as the first known PP1 regulator that suppresses catalytic activity by conformationally locking an active-like state, rather than by disrupting the active site or occluding substrate-binding grooves. By sequestering PP1 in a primed yet silenced state, SCRIB establishes a latent cortical pool that provides a molecular mechanism for rapid, localized phosphatase activation following upstream stimuli.