Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.     

Non-catalytic domains of subunit A negatively regulate the activity of calcineurin

Calcineurin is composed of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). In addition to the catalytic core, CNA further contains three non-catalytic domains--CNB binding domain (BBH), calmodulin binding domain (CBD), and autoinhibitory domain (AI). To investigate the effect of these three domains on the activity of CNA, we have constructed domain deletion mutants CNAa (catalytic domain only), CNAac (CNAa and CBD), and CNAaci (CNAa, CBD and AI). By using p-nitrophenylphosphate and (32)P-labeled R(II) peptide as substrates, we have systematically examined the phosphatase activities, kinetics, and regulatory effects of Mn(2+)/Ni(2+) and Mg(2+). The results show that the catalytic core has the highest activity and the order of activity of the remaining constructs is CNAac>CNAaci>CNA. Sequential removal of the non-catalytic domains corresponds to concurrent increases of the phosphatase activity assayed under several conditions. This observation clearly demonstrates that non-catalytic domains negatively regulate the enzyme activity and act as intra-molecular inhibitors, possibly through restraining the conformation elasticity of the catalytic core required for optimal catalysis or interfering with substrate access. The sequential domain deletion favors activation of the enzyme by Mn(2+)/Ni(2+) but not by Mg(2+) (except for CNAa), suggesting that enzyme activation by Mn(2+)/Ni(2+) is mainly mediated via the catalytic domain, whereas activation by Mg(2+) is via both the catalytic core and non-catalytic domains.     

A renewed model of CNA regulation involving its C-terminal regulatory domain and CaM

Calcineurin is composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). CNA contains the catalytic domain and three regulatory domains: a CNB-binding domain (BBH), a C-terminal calmodulin-binding domain (CBD), and an autoinhibitory domain (AID). We constructed a series of mutants of CNA to explore the regulatory role of its C-terminal regulatory domain and CaM. We demonstrated a more precise mechanism of CNA regulation by C-terminal residues 389-511 in the presence of CNB. First, we showed that residues 389-413, which were identified in previous work as constituting a CaM binding domain (CBD), also have an autoinhibiting function. We also found that residues 389-413 were not sufficient for CaM binding and that the CBD comprises at least residues 389-456. In conclusion, two distinct segments of the C-terminal regulatory region (389-511) of CNA inhibit enzyme activity: residues 389-413 interact with the CNB binding helix (BBH), and residues 457-482 with the active center of CNA.     

Four loops of the catalytic domain of factor viia mediate the effect of the first EGF-like domain substitution on factor viia catalytic activity

The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca(2+)-bound factor VIIa and FVII((IXegf1))a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII((IXegf1))a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A+R147A+D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170s(del). The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.     

一种新的人CNA1基因转录本的克隆和功能鉴定

钙调神经磷酸酶（calcineurin,CN）是唯一依赖于Ca2+和钙调蛋白（calmodulin,CaM）的丝氨酸/苏氨酸型蛋白磷酸酶,由1个催化亚基CNA和1个调节亚基CNB组成. CNA 有3种亚型,最常见的是由CNA1基因编码的α亚型（CNAα）. 在克隆CNA1基因cDNA的过程中,发现了1种新的人CNA1转录本-CNAα4. 与CNA1基因的其它转录本相比,CNAα4缺失第2外显子,其编码蛋白质由454个氨基酸组成,具有比其它3种CNAα亚型更短的磷酸酶催化结构域. CNAα4具有与CNAα1相似的CaM亲和力,但是其激活活化T细胞核因子（nuclear factor of activated T cells,NFAT）的活性明显强于CNAα1,提示CNAα4所缺失的氨基酸序列（Ala20 Thr86）并非CNA催化结构域所必需,相反,Ala²⁰-Thr⁸⁶缺失可能有助于其酶活性中心与NFAT的结合并发挥作用.     

The regulatory domains of CNA have different effects on the inhibition of CN activity by FK506 and CsA

Calcineurin (CN) is the common receptor for two immunophilin-immunosuppressant complexes, Cyp-CsA and FKBP-FK506. Calcineurin is composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). CNA contains the catalytic domain and three regulatory domains: a CNB-binding domain (BBH, 350-370), a calmodulin- binding domain (CBD, 389-413), and an autoinhibitory domain (AID, 457-482). To investigate the effects of these three regulatory domains on the inhibition of CN by the two drugs we constructed three C-terminal deletion mutants: CNAabc (1-456), CNAab (1-388) and CNAa (1-347). Inhibition of CNA and its derivatives by the two drugs was examined and compared with inhibition by peptides (AID [457-482] and LCBD [389-456], CBD and the extension of the AID were included). Our results show that the BBH is critical for inhibition of CN by Cyp-CsA and FKBP-FK506. The LCBD has no effect and the AID reduces the inhibition of CN by two complexes. In addition, LCBD and AID as autoinhibitors may inhibit enzyme activity via different sites.     

Function and structure of N-terminal and C-terminal domains of calcineurin B subunit

Calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, plays a critical role in T-cell activation by regulating the activity of NF-AT. CN is a heterodimer consisting of a catalytic subunit (CNA) and a Ca2+-binding regulatory subunit (CNB). CNB is composed of two global domains: the C-terminal domain (DC) and the N-terminal domain (DN), each containing two Ca2+ binding sites. In this study, using purified DN and DC derived from constructed expression systems, we revealed that intact CNB and DC can stimulate the phosphatase activity of CNA, about 2.2 and 1.6 times the phosphatase activity of CNA alone, respectively; DN itself has little effect on the phosphatase activity of CNA. Fluorescence spectroscopy of an ANS-hydrophobic fluorescence probe shows that binding of Ca2+ to CNB, DC or DN leads to exposure of the hydrophobic surface of the proteins and that the hydrophobicity of CNB is the greatest, that of DC is less, and that of DN is the least. The hydrophobic surface of CNB may be an important structural basis for stimulating CN phosphatase activity.     

The role of loop 7 in mediating calcineurin regulation

Calcineurin (CN) is a heterodimer protein consisting of a 61 kDa catalytic subunit A and a 19 kDa regulatory subunit B. It plays a critical role in T-cell activation and is involved in many cellular processes. Regulation of CN is rather complex, including a number of factors such as divalent metal ions (primarily Ca(2+) and Mn(2+)), calmodulin (CaM) and autoinhibition (AI) segment. Previously, we reported that a loop 7 deletion mutant (V314) in subunit A exhibited high phosphatase activity, although the mechanism for the surprising activity enhancement and whether the activity change applies to other loop 7 residues were not known. In order to probe the role of loop 7, we have carried out extensive mutagenesis experiments, followed by systematic activity assays under a number of regulatory conditions. All mutants, including single deletion mutants Y315, N316 and double deletion mutant V314Y315, showed increased phosphatase activity. Significantly, activities of the mutants containing the V314 deletion, namely V314 and V314Y315, were no longer regulated by regulatory subunit B. These results, along with the structure analysis, suggest that loop 7 as a whole plays an important role in mediating CN's regulation through bridging the regulatory subunit and catalytic core and interaction with the AI segment of CN.     

Preparation and characterization of a single-chain calcineurin-calmodulin complex

Calcineurin (CN), a Ca(2+)/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca(2+)-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.     

The beta12-beta13 loop is a key regulatory element for the activity and properties of the catalytic domain of protein phosphatase 1 and 2B

The molecular architectures of the catalytic core of protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) are similar, and both contain a beta12-beta13 loop that consists of non-conserved residues. A truncation mutant containing the PP2B catalytic domain has previously been constructed in our laboratory, and designated CNAa. In this study, the PP1 catalytic subunit (PP1c) and CNAa, as well as mutants with the corresponding loops exchanged, were investigated using multiple substrates. Deletion of the beta12-beta13 loop from Y272 to A279 of PP1c or from Y311 to K318 of CNAa resulted in inactive proteins. Loop exchange generated chimeric mutants called PP1-CNAa-loop and CNAa-PP1-loop. The activities and kinetic parameters of the two chimeric mutants were altered in the direction of the enzyme from which its loop was derived. The activity of PP1c or CNAa-PP1-loop was similar whether preincubated with Mn(2+) or not, while CNAa and PP1-CNAa-loop can acquire enhanced activation if preincubated with Mn(2+) for longer periods of time. Intrinsic fluorescence spectra revealed that the three-dimensional structure was altered as a result of exchanging the loops of PP1c and CNAa. In conclusion, the beta12-beta13 loop is one of the key regulatory elements in the catalytic domain for the activity and properties of PP1c and CNAa.     

Kaempferol: a new immunosuppressant of calcineurin

Calcineurin (CN), the Ca(2+)/calmodulin (CaM)-dependant protein phosphatase, is the target for immunosuppressive drugs cyclosporine A (CsA) and FK506. These immunosuppressants can inhibit CN activity after binding with respective immunophilins. Based on the model of screening by using p-nitrophenyl phosphate as a substrate for preliminary screening and (32)P-labeled 19-residue phosphopeptide as a specific substrate for final determination, we found Kaempferol, a natural flavonol, could inhibit CN activity in purified enzyme and Jurkat T-cells. Unlike CsA and FK506, CN inhibition by kaempferol is independent of matchmaker protein and the inhibitory manner is noncompetitive. Through investigation of inhibitions for CNA and a series of its truncated mutants, we suggested that Kaempferol could directly act on the catalytic domain. Data also indicated that the CN inhibition by kaempferol could be enhanced when the enzyme was activated in the presence of CaM and CNB. CNB is necessary for mediating inhibition of enzyme by kaempferol. The result of RT-PCR also indicated that kaempferol had an inhibitory activity against IL-2 gene expression in activated Jurkat cells. All data suggested that kaempferol could be a new immunosuppressant of CN.     

Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase

The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp-392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu.     

The catalytically active domain in the A subunit of calcineurin

Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAdelta that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn2+ and Ni2+ ions effeciently activated CaNA. The Km of a/CaNA was about 16 mM, and the k(cat) of a/CaNA was 10.03 s(-1) using pNPP as substrate. With RII peptide as a substrate, the Km of a/CaNA was about 21 microM and the k(cat) of a/CaNA was 0.51 s(-1). The optimum reaction temperature was about 45 degrees C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-subunit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN.     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis.Their phosphatase activity and the corresponding solution conformation were examined.Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immunosuppressive drugs with CN.The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN.Furthermore,circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein.Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN,and had no effects on the phosphatase activity of mutants in Loop7 region,which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN.Examination of the activities of these mutants resulted in the presence of immunosuppressive component from traditional Chinese drugs.The component of Chinese drug,ZIP1,could directly inhibit both CN and CN mutants without drug binding protein.These results suggest that the Loop7 region is an important structural area involved in the inhibition by CyP-CsA.It is valuable to further study the inhibition by ZIP1.     

The salt bridge of calcineurin is important for transferring the effect of CNB binding to CNA

Calcineurin (CN) is a heterodimer consisting of a catalytic subunit (CNA) and a regulatory subunit (CNB). The crystal structure shows that three residues or regions of CNA are mainly responsible for the interaction with CNB: the CNB binding helix (BBH), the N-terminus, and Glu53 that forms a salt bridge with Lys134 of CNB. In this report, we try to find the role that the salt bridge plays in the interaction between CNA and CNB. We found that mutation of Glu53 greatly reduced its responsiveness to CNB in the phosphatase assay and also that mutation of Lys134 of CNB affected its ability to activate the phosphatase activity of CNA. Structural analysis showed that disruption of the salt bridge affected the compact association of CNA and CNB. Thus, the salt bridge appears to help to stabilize CN and transfer the effects of CNB binding to CNA to activate its phosphatase activity.     

Structural basis for substrate recognition and hydrolysis by mouse carnosinase CN2

L-carnosine is a bioactive dipeptide (beta-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn(2+), and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn(2+) at a resolution of 1.7 A and that with Mn(2+) at 2.3 A CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn(2+) and Zn(2+) complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.     

Characterization of the fragmented forms of calcineurin produced by calpain I

Calcineurin, a calmodulin-stimulated phosphatase from bovine brain, was hydrolyzed by calpain I from human erythrocytes. In the absence of calmodulin, calpain rapidly transformed the 60-kilodalton (kDa) catalytic subunit of calcineurin into a transient 57-kDa fragment and thereafter a 43-kDa limit fragment. In the presence of calmodulin, the 60-kDa subunit was sequentially proteolyzed to a 55-kDa fragment and then a 49-kDa fragment. Upon proteolysis in the absence or presence of calmodulin, the p-nitrophenyl phosphatase activity (assayed in the presence of calmodulin) was increased by 300%. The 43- and the 49-kDa fragments were found to (i) remain associated with the small subunit (17 kDa), (ii) have lost the ability to bind and to be activated by calmodulin, and (iii) have phosphatase activity that was still stimulated by Mn2+ or Ni2+. The 43- + 17-kDa form had similar Km values for various substrates, but the Vmax values were increased compared with the native enzyme. It is proposed that (i) a 43-kDa core segment of the 60-kDa subunit of calcineurin contained the catalytic domain, the small subunit-binding domain, and the metal ion (Mn2+ and (or) Ni2+) binding site; and (ii) two distinct types of inhibitory domains exist near the end(s) of the large subunit, one of which is calmodulin regulated, while the other is calmodulin independent.     

Effect of vanadyl ions on calcineurin and its A subunit

Calcineurin (CN) exhibits a bimodal regulation by different concentrations of vanadyl ions (VO2+) in the presence of Mn2+. Low concentrations of VO2+ inhibit the enzyme, with 50 microM VO2+ completely inhibiting CN activity, while high concentrations, up to 500 microM VO2+, stimulate the CN activity. A similar bimodal regulation of CN was not observed with either calcium or vanadate under the same conditions. X-band electron spin resonance spectroscopy, used to study the binding of VO2+ to the catalytic subunit A of calcineurin, show that there are two kinds of binding sites in the A subunit.     

Interaction of glycyrol with calcineurin A studied by spectroscopic methods and docking

We have shown previously that glycyrol has an inhibitory effect on the immune response in mice by reducing calcineurin activity (Li et al., 2010, Pharm Biol 48:1177–1184). Here, we investigated the interaction of glycyrol with calcineurin A (CNA, catalytic A subunit of calcineurin) by spectroscopic methods and docking. We showed that glycyrol binds to CNA via hydrophobic interactions in a ratio of 1:1, and the main binding site is in the catalytic domain of CNA close to the calcineurin B subunit-binding domain. Binding of glycyrol changes the secondary structure of CNA, and this effect may possibly inhibit CN activity.