“…In addition, many of the peaks originating from the 27 residues that connect the NHE1 LxVP and PxIxIT motifs were also broadened upon complex formation (Fig. 2a, b), indicating either that this region is involved in the interaction with CN or that the conformational freedom of the linker is impaired by the anchoring of both SLiMs to CN 24,25 . Thus, NHE1 684 LTVP 687 is a CN-specific LxVP SLiM that, together with NHE1 715 PVITID 720 , binds to CN.Fig.…”
Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na
+
/H
+
-exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer
p
Thr over
p
Ser, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of
p
T779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.
“…In addition, many of the peaks originating from the 27 residues that connect the NHE1 LxVP and PxIxIT motifs were also broadened upon complex formation (Fig. 2a, b), indicating either that this region is involved in the interaction with CN or that the conformational freedom of the linker is impaired by the anchoring of both SLiMs to CN 24,25 . Thus, NHE1 684 LTVP 687 is a CN-specific LxVP SLiM that, together with NHE1 715 PVITID 720 , binds to CN.Fig.…”
Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na
+
/H
+
-exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer
p
Thr over
p
Ser, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of
p
T779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.
“…Unfortunately, MCs have not so far yielded suitable crystals for such studies. However, in recent decades the 3-dimensional structures of MCs bound to protein phosphatases (PPs) have been determined [118][119][120][121][122][123][124][125][126] (see Section 6.2). In addition to providing key information about the mode of action and structure-activity relationships of the MCs, this also provides 3-dimensional structural information about the toxins themselves.…”
Section: -Dimensional Structures Of Microcystinsmentioning
Hepatotoxic microcystins (MCs) are the most widespread class of cyanotoxins and the one that has most often been implicated in cyanobacterial toxicosis. One of the main challenges in studying and monitoring MCs is the great structural diversity within the class. The full chemical structure of the first MC was elucidated in the early 1980s and since then, the number of reported structural analogues has grown steadily and continues to do so, thanks largely to advances in analytical methodology. The structures of some of these analogues have been definitively elucidated after chemical isolation using a combination of techniques including nuclear magnetic resonance, amino acid analysis, and tandem mass spectrometry (MS/MS). Others have only been tentatively identified using liquid chromatography-MS/MS without chemical isolation. An understanding of the structural diversity of MCs, the genetic and environmental controls for this diversity and the impact of structure on toxicity are all essential to the ongoing study of MCs across several scientific disciplines. However, because of the diversity of MCs and the range of approaches that have been taken for characterizing them, comprehensive information on the state of knowledge in each of these areas can be challenging to gather. We have conducted an in-depth review of the literature surrounding the identification and toxicity of known MCs and present here a concise review of these topics. At present, at least 279 MCs have been reported and are tabulated here. Among these, about 20% (55 of 279) appear to be the result of chemical or biochemical transformations of MCs that can occur in the environment or during sample handling and extraction of cyanobacteria, including oxidation products, methyl esters, or post-biosynthetic metabolites. The toxicity of many MCs has also been studied using a range of different approaches and a great deal of variability can be observed between reported toxicities, even for the same congener. This review will help clarify the current state of knowledge on the structural diversity of MCs as a class and the impacts of structure on toxicity, as well as to identify gaps in knowledge that should be addressed in future research.
“…1 A and B, Top) (4,7). Although the specificity of PPPs appears low, PP1 is nevertheless able to dephosphorylate its targets with high specificity (8). This specificity is achieved via its interaction with >200 known regulatory proteins that bind directly to PP1, targeting it to specific cellular locations and substrates (9)(10)(11).…”
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.
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