SDS22 selectively recognizes and traps metal-deficient inactive PP1

Abstract

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.

Protein phosphatase 1: life‐course regulation by SDS22 and Inhibitor‐3

“…This article is protected by copyright. All rights reserved [30] rather suggest that SDS22 is recruited to pre-existing pools of inactive PP1. In conclusion, although a high SDS22:I3 ratio clearly reduces the activity of PP1 in intact cells, it is uncertain whether such inhibitory SDS22:I3 ratios ever occur in vivo.…”

“…Inactivation blocks the activity towards all substrates and renders the catalytic core of PP1 sensitive to trypsinolysis, which can be explained by the loss of catalytic-site metal(s) and/or a conformational change in the catalytic core [33]. Hence, inactive PP1:SDS22 is likely to be identical to the crystallized heterodimer, in which PP1 lacks metal 1 and is conformationally altered [30] (Fig. 3B).…”

“…It is surprising that the extraction of I3 from SDS22:PP1:I3 requires ATP hydrolysis as the interaction between PP1 and SLiM-based RIPPOs, such as I3, is highly dynamic and allows a spontaneous exchange of PP1, albeit possibly at lower rates [30,46]. Also, the need to 'unfold' I3 in the p97 hexameric channel is difficult to grasp as free I3 has no stable secondary of tertiary Accepted Article structure.…”

“…with I3, possibly aided by their direct interaction [24]. Alternatively, the extraction of SDS22 may be coupled to conformational changes of PP1, associated with metal loading, which reduces its affinity for SDS22 [30].…”

Molecular basis for Ras suppressor-1 recruitment to focal adhesions and stabilization of consensus adhesome complex

“…The copyright holder for this preprint this version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.19.432017 doi: bioRxiv preprint 7 the SDS22-like subfamily from the DALI search is the extracellular domain of mouse platelet receptor glycoprotein Ib (GPIb) (26). Those two representative SDS22 class LRR proteins bind their cognate partner proteins through their concave surfaces (27,28). Thus, it is of interest to understand how Rsu-1 with similar concave surface recognizes a different target (see below).…”