The protein serine/threonine phosphatase calcineurin (CN) is activated by calmodulin (CaM) in response to intracellular calcium mobilization. A widely accepted model for CN activation involves displacement of the CN autoinhibitory peptide (CN [467][468][469][470][471][472][473][474][475][476][477][478][479][480][481][482][483][484][485][486] ) from the active site upon binding of CaM. However, CN activation requires calcium binding both to the low affinity sites of CNB and to CaM, and previous studies did not dissect the individual contributions of CNB and CaM to displacement of the autoinhibitory peptide from the active site. In this work we have produced separate CN fragments corresponding to the CNA regulatory region (CNRR 381-521 , residues 381-521), the CNA catalytic domain truncated at residue 341, and the CNA-CNB heterodimer with CNA truncated at residue 380 immediately after the CNB binding helix. We show that the separately expressed regulatory region retains its ability to inhibit CN phosphatase activity of the truncated CN341 and CN380 and that the inhibition can be reversed by calcium/CaM binding. Tryptophan fluorescence quenching measurements further indicate that the isolated regulatory region inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The results provide new support for a model in which calcium binding to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. Moreover, the secondary structural content of the CNRR 381-521 was tentatively addressed by Fourier transform infrared spectroscopy. The results indicate that the secondary structure of CNRR 381-521 fragment is predominantly random coil, but with significant amount of ␤-strand and ␣-helix structures. Calcineurin (CN),3 also called protein phosphatase 2B, is a calcium/CaM-dependent Ser/Thr protein phosphatase (1-3) and plays a critical role in the coupling of Ca 2ϩ signals to cellular responses (3-10). CN is stimulated by the multifunctional protein, calmodulin (CaM), which ensures the coordinated regulation of CN protein phosphatase activity, together with the activities of many other enzymes, including a large number of protein kinases under Ca 2ϩ and CaM control (7). CN has a wide range of physiological substrates (7). The complex regulation observed with CN is expected for an enzyme that is a major player in the regulation of many cellular processes. Various phosphoproteins such as inhibitor 1a, protein kinase A regulatory subunit RII, neurogranin, phosphorylase kinase a, neuromodulin, and small organic substrate p-nitrophenyl phosphate are all dephosphorylated by CN (7). Among CN substrates, the nuclear factor of activated T cells (NFAT) family of transcription factors is arguably the best understood (2, 9). NFAT is a phosphoprotein located in the cytoplasm of the resting cell. In response to physiological signals that elevate intracellular calcium, NFAT ...

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The Escherichia coli cyclic AMP receptor protein (CRP) is a well known transcription activator protein. In this study, CRP was overexpressed, purified and cocrystallized with cAMP and a 38 bp full-length double-stranded DNA fragment. The full-length segment differed from the half-site fragments used in previous crystallization experiments and is more similar to the environment in vivo. CRP-cAMP-DNA crystals were obtained and diffracted to 2.9 Å resolution. The crystals belonged to space group P3 1 21, with unit-cell parameters a = b = 76.03, c = 144.00 Å . The asymmetric unit was found to contain one protein molecule and half a 38 bp full-length double-stranded DNA fragment, with a Matthews coefficient of 2.62 Å 3 Da À1 and a solvent content of 53.14%.

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