The structure of the RCAN1:CN complex explains the inhibition of and substrate recruitment by calcineurin

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the Ser/Thr phosphatase calcineurin (CN). It has been shown that excessive inhibition of CN is a critical factor for Down syndrome and Alzheimer's disease. Here, we determined RCAN1's mode of action. Using a combination of structural, biophysical, and biochemical studies, we show that RCAN1 inhibits CN via multiple routes: first, by blocking essential substrate recruitment sites and, second, by blocking the CN active site using two distinct mechanisms. We also show that phosphorylation either inhibits RCAN1-CN assembly or converts RCAN1 into a weak inhibitor, which can be reversed by CN via dephosphorylation. This highlights the interplay between posttranslational modifications in regulating CN activity. Last, this work advances our understanding of how active site inhibition of CN can be achieved in a highly specific manner. Together, these data provide the necessary road map for targeting multiple neurological disorders.National Institute of General Medical SciencesOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Nuclear Magnetic Resonance Structure and Dynamics of the Response Regulator Sma0114 from <i>Sinorhizobium meliloti</i>

Receiver domains control intracellular responses triggered by signal transduction in bacterial two-component systems. Here, we report the solution nuclear magnetic resonance structure and dynamics of Sma0114 from the bacterium <i>Sinorhizobium meliloti</i>, the first such characterization of a receiver domain from the HWE-kinase family of two-component systems. The structure of Sma0114 adopts a prototypical α₅/β₅ Rossman fold but has features that set it apart from other receiver domains. The fourth β-strand of Sma0114 houses a PFxFATGY sequence motif, common to many HWE-kinase-associated receiver domains. This sequence motif in Sma0114 may substitute for the conserved Y-T coupling mechanism, which propagates conformational transitions in the 455 (α4−β5−α5) faces of receiver domains, to prime them for binding downstream effectors once they become activated by phosphorylation. In addition, the fourth α-helix of the consensus 455 face in Sma0114 is replaced with a segment that shows high flexibility on the pico- to nanosecond time scale by ¹⁵N relaxation data. Secondary structure prediction analysis suggests that the absence of helix α4 may be a conserved property of the HWE-kinase-associated family of receiver domains to which Sma0114 belongs. In spite of these differences, Sma0114 has a conserved active site, binds divalent metal ions such as Mg²⁺ and Ca²⁺ that are required for phosphorylation, and exhibits micro- to millisecond active-site dynamics similar to those of other receiver domains. Taken together, our results suggest that Sma0114 has a conserved active site but differs from typical receiver domains in the structure of the 455 face that is used to effect signal transduction following activation

Molecular basis for the binding and selective dephosphorylation of Na+/H+ exchanger 1 by calcineurin

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 pThr over pSer, 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 pT779. 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.Lundbeck Foundation; National Institute of Neurological Disorders and Stroke [R01NS091336]; National Institute of General Medicine [R01GM098482]; Danish Council for Independent Research Natural Sciences [4181-00344]; Novo Nordisk Foundation [NNF18OC0034070]; US Department of Energy, Office of Science and Office of Basic Energy Sciences [DE-AC02-76SF00515]; DOE Office of Biological and Environmental Research; National Institutes of Health, National Institute of General Medical Sciences [P41GM103393]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

pH Dependence of Amylin Fibrillization

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic β-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has two ionizable residues between pH 3 and 9: the α-amino group and His18. Our approach to measuring the p<i>K</i>_a values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the two groups. The α-amino group at the unstructured N-terminus of amylin has a p<i>K</i>_a near 8.0, similar to the value in random coil models. By contrast, His18, which is involved in the intermolecular β-sheet structure of the fibrils, has a p<i>K</i>_a that is lowered to 5.0 in the fibrils compared to the random coil value of 6.5. The lowered p<i>K</i>_a of His18 is due to the hydrophobic environment of the residue, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. His18 acts as an electrostatic switch inhibiting fibrillization in its charged state. The presence of a charged side chain at position 18 also affects fibril morphology and lowers amylin cytotoxicity toward a MIN6 mouse model of pancreatic β-cells. In addition to the two expected p<i>K</i>_a values, we detected an apparent p<i>K</i>_a of ∼4.0 for the amylin-derived peptide NAc-SNN­F­G­A­ILSS-NH₂, which has no titratable groups. This p<i>K</i>_a is due to the pH-induced ionization of the dye thioflavin T. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry, we were able to distinguish between the titration of the dye and groups on the peptide. Large differences in reaction kinetics were observed between the different methods at acidic pH, because of charges on the ThT dye, which hinder fibril formation much like the charges on the protein