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The Golgi PMR1 P-type ATPase of Caenorhabditis elegans. Identification of the gene and demonstration of calcium and manganese transport.

In recent years, it has been well established that the Ca(2+) concentration in the lumen of intracellular organelles is a key determinant of cell function. Despite the fact that essential functions of the Golgi apparatus depend on the Ca(2+) and Mn(2+) concentration in its lumen, little is known on the transport system responsible for ion accumulation. The Golgi ion pump PMR1 has been functionally studied only in yeast. In humans, mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. The C. elegans genome is predicted to contain a single PMR1 orthologue on chromosome I. We found evidence for alternative splicing in the 5'-untranslated region, but no indication for the generation of different protein isoforms. C. elegans PMR1 overexpressed in COS-1 cells transports Ca(2+) and Mn(2+) with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca(2+) can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca(2+) store.

In eukaryotic cells, cytosolic Ca 2ϩ acts as a second messenger in a large variety of cell functions. The increase of the cytosolic free Ca 2ϩ concentration in response to a stimulus results from the opening of Ca 2ϩ channels present in the plasma membrane and in the membranes of intracellular Ca 2ϩ stores, mainly the endoplasmic reticulum (ER). 1 It has more recently also become clear that the Ca 2ϩ stored in the lumen of many intracellular compartments not only serves as a reservoir of releasable Ca 2ϩ , but also plays a regulatory role in several important cell biological functions. Luminal Ca 2ϩ in the ER as well as in the Golgi or other components of the secretory pathway is required for the proper translation, translocation, folding, and processing of secreted proteins (for review, see Ref. 1). A sufficiently high level of intraorganellar Ca 2ϩ has been implicated also in intra-Golgi membrane transport (2), transport between the Golgi and the ER (3), and in endosome fusion (4). Besides these constitutive actions, luminal Ca 2ϩ controls movements of the Ca 2ϩ ion itself: its release from the intracellular stores, its permeability through the nuclear pore complexes, and capacitative Ca 2ϩ entry at the plasma membrane (for review, see Ref. 5).
The pivotal role of the ER as a Ca 2ϩ store is well established. There is a growing consensus that also the Golgi apparatus can function as an agonist-releasable Ca 2ϩ store (6), the importance of which may, however, greatly differ among the different cell types and ranges from relatively unimportant in Drosophila melanogaster S2 cells (7) to very important in renal LLC-PK 1 cells (8). The storage capacity for Ca 2ϩ in the Golgi is greatly increased by specific proteins like Calnuc/nucleobindin (9,10) and members of the CREC family of Ca 2ϩ -binding proteins (reviewed in Ref. 11). In contrast to the growing unanimity on the role of Golgi as a Ca 2ϩ store, there remains some confusion as to the type of Ca 2ϩ -accumulation system responsible to replenish the store. Ca 2ϩ pumps belonging to the class of P-type ion-motive ATPases have been proposed to take up this task: SERCA2 (6,10), the plasma-membrane Ca 2ϩ pump PMCA en route to the plasma membrane (12) and PMR1 (6,13). The SERCA pumps, which are encoded by three different genes in vertebrates (human gene names: ATP2A1-3), but apparently only by a single gene in invertebrates like C. elegans, are the best characterized members of this class and are responsible for the accumulation of Ca 2ϩ into the ER or into the sarcoplasmic reticulum. The PMCA Ca 2ϩ pumps (corresponding human gene names: ATP2B1-4) responsible for the extrusion of Ca 2ϩ out of the cell, also belong to the same class but are themselves not involved in Ca 2ϩ transport into the stores, with as a possible exception the above mentioned Golgi-based PMCA pumps which are on their way to the plasma membrane. The PMR1-type of Ca 2ϩ -transport ATPases was first identified in the yeast Saccharomyces cerevisiae (14) and localized to the Golgi or one of its subcompartments (15). Genes homologous to the S. cerevisiae PMR1 have been reported for a number of other fungi (see Ref. 16 and references therein). The PMR1 ion-motive ATPase supplies the secretory pathway with Ca 2ϩ and Mn 2ϩ ions required for glycosylation, sorting, and ERassociated protein degradation (17,18). A recent study has demonstrated capacitative Ca 2ϩ entry in S. cerevisiae, a mechanism that in higher eukaryotes is thought to be initiated by depletion of intracellular stores that are filled by the SERCA Ca 2ϩ pump (19). The process was stimulated in pmr1 mutants, indicating that in yeast capacitative Ca 2ϩ entry in combination with PMR1 activity supplies the secretory pathway with Ca 2ϩ . Yeast pmr1 mutants do not grow on a medium containing submicromolar concentrations of Ca 2ϩ and show defects in the maturation of secretory proteins which are suppressed by supplying millimolar Ca 2ϩ or micromolar Mn 2ϩ to the growth medium. Mutations in PMR1 were also reported to rescue yeast mutants, which as a result of the lack of superoxide dismutase, show impaired growth in aerobic conditions (20). This effect is ascribed to increased cytosolic levels of Mn 2ϩ resulting from a lack of accumulation of the ion in the Golgi compartment. Mn 2ϩ is known for its capacity to scavenge superoxide ions. Mandal et al. (21) pointed recently to the critical role of transmembrane segment M6 in yeast PMR1 for defining the cation-binding sites in general and in particular of residue Gln 783 in this segment for the Mn 2ϩ selectivity.
Relatively less is known on the PMR1 homologues in animal cells. The cDNA of the putative rat form of the yeast PMR1 was already cloned in 1992 with a SERCA-derived probe (13), but the authors failed in their efforts to show that the corresponding protein, upon its expression in COS cells, was able to catalyze the uptake of Ca 2ϩ into vesicles consisting of fragmented membranes. In the meanwhile homologous cDNAs or genes were reported for D. melanogaster, 2 Bos taurus, 3 and C. elegans. But again until now no direct indication that any of these was involved in Ca 2ϩ uptake has been provided. However, indirect evidence comes from two recent reports which show that Hailey-Hailey disease (MIM 16960), which is manifested by the impaired intercellular adhesion of epidermal keratinocytes, results from mutations in one of the alleles of a human orthologue of yeast PMR1, the ATP2C1 gene (22,23). The symptoms of Hailey-Hailey disease strongly resemble those of Darier-White disease (MIM 124200) which is due to a mutation in one of the alleles of the SERCA2 gene ATP2A2. This, together with the observation that expression of the mammalian SERCA1a prevented the lethality of the pmr1-pmc1 double mutations in yeast (24), strongly suggests that the human PMR1 pump can act as a Ca 2ϩ pump.
We now show for the first time that an animal PMR1 homologue can transport Ca 2ϩ or Mn 2ϩ into the Golgi apparatus of COS-1 cells with high affinity and in a thapsigargin-insensitive manner. The accumulated Ca 2ϩ can be released by IP 3 , in line with the view that the Golgi apparatus is an IP 3 -sensitive Ca 2ϩ store.

EXPERIMENTAL PROCEDURES
Materials-[␥-32 P]ATP and 45 Ca were obtained from Amersham Pharmacia Biotech (Roosendaal, The Netherlands), 54 Mn from PerkinElmer Life Sciences (Boston, MA). Sequencing was done with the AutoRead 200 Sequencing Kit from Amersham Pharmacia Biotech. COS-1 cells were transiently transfected with FuGENE 6 transfection reagent (Roche Molecular Diagnostics, Brussels, Belgium) according to the manufacturer's instructions. Fluorescein isothiocyanate-labeled goat anti-rabbit antibodies were obtained from Sigma (Sigma/Aldrich NV, Bornem, Belgium) and horseradish peroxidase-labeled swine antirabbit antibodies from Dako (Dako A/S, Glostrup, Denmark). The fulllength rabbit SERCA1a clone was kindly provided by J. P. Andersen and B. Vilsen (University of Aarhus, Denmark).
Constructs for Expression-The EST clones yk218a11 and yk334d5 were obtained from Yuji Kohara's database, and sequenced. Both clones contained the complete open reading frame of C. elegans PMR1. It should, however, be remarked that the open reading frame is 63 bp smaller than predicted in the database annotation (see "Results"). Clone yk334d5 was used to make an expression construct. The 3.2-kb PMR1-encoding EcoRI/XhoI fragment of yk334d5 was ligated into the dephosphorylated pcDNA3 expression vector (Invitrogen Co., British Biotechnology Products Ltd., Abingdon, United Kingdom) cut with the same restriction enzymes. The pig SERCA2a expression vector has been described by Verboomen et al. (25). The full-length rabbit SERCA1a was cloned in the pMT2 expression vector (26).
Cell Culture and DNA Transfection-For microsome preparation, COS-1 cells were seeded in 100-mm culture dishes at a density of 2.5 ϫ 10 6 cells per plate. For immunocytochemistry 2.0 ϫ 10 4 cells were seeded on gelatin (1%)-coated coverslips. For 45 Ca 2ϩ or 54 Mn 2ϩ fluxes 3.0 ϫ 10 4 cells were seeded in gelatin-coated 12-well plates. For microsome preparation and immunocytochemistry, transfection was performed the day after seeding. For isotope fluxes the period between seeding and transfection was extended to 5 days to allow better attachment of the cells to the plates. After transfection, the cells were incubated for 60 h at 37°C and 5% CO 2 .
Preparation of Antiserum to PMR1 Protein-The immunogen was a recombinant protein corresponding to the putative large cytoplasmic loop between transmembrane segments 4 and 5 of C. elegans PMR1. The protein was expressed in Escherichia coli using the QIAexpress Type IV System (Qiagen, Hilden, Germany). In a first step the cDNA corresponding to the loop region was amplified by PCR with primers PMR1CYTF (5Ј-CAAGCATGCCGTGAAGAAGATGCCAGCAG-3Ј) and PMR1CYTR (5Ј-CTAGTCGACTTTTCCCTCCTCAATCGCC-3Ј) containing, respectively, a SphI and SalI restriction site at their 5Ј end. The PMR1CYTF primer corresponds to nucleotides 26812-26831 of cosmid CECC4 (accession number Z81490) and PMR1CYTR primer to the inverse complement of nucleotides 136 -154 of cosmid CEZK256 (accession number Z82088). PCR was carried out for 20 cycles using the Expand Long Template PCR System from Roche Molecular Diagnostics. Each cycle consisted of 10 s of denaturation at 94°C, 30 s of annealing at 55°C, and 2 min of elongation at 68°C. The SphI/SalI-cut PCR fragment was ligated in the SphI/SalI sites of the pQE-31 bacterial expression vector (Qiagen), containing a 6xHis tag coding sequence 5Ј to the cloning region. The expression of the recombinant protein in E. coli M15 cells was induced by 1 mM isopropyl-1-thio-␤-D-galactopyranoside and purification of the 6xHis-tagged protein was achieved by a Ni-NTA agarose-based method as described in the manufacturer's instructions. The recombinant protein migrated with an apparent molecular mass of about 42 kDa in 12% SDS-polyacrylamide gels and reacted with the monoclonal anti-polyhistidine antibody (clone HIS-1, from Sigma, dilution 1:1000) on Western blots. In a last step the recombinant cytoplasmic loop was concentrated by Centricon TM Plus-20 centrifugal filtering (Millipore, Bedford, MA).
Rabbits were immunized with 0.1 mg of recombinant protein in 0.5 ml of phosphate-buffered saline emulsified with 0.5 ml of complete Freund's adjuvant. Booster injections of the same immunogen with incomplete Freund's adjuvant were given at 4-week intervals. Preimmune serum and serum obtained after 4 boosters were used. The antiserum is designated as Celpmrloop.
Membrane Preparations and Immunoblotting Analysis-Microsomes were isolated from COS-1 cells as described by Verboomen et al. (25). Membranes from C. elegans were isolated as described by Baylis et al. (28). Protein concentrations were determined by the bicinchoninic acid method (Pierce, Rockford, IL). Denaturing gel electrophoresis and Western blotting were done as described earlier (29). 45 Ca 2ϩ / 54 Mn 2ϩ Fluxes-COS-1 cells were grown on 12-well plates. Loading with 45 Ca 2ϩ and efflux were done essentially as described earlier (30). Cells were treated for 10 min with 20 g/ml saponin at 25°C and loaded for the indicated lengths of time in 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM ATP, 10 mM NaN 3 , 0.44 mM (for Ca 2ϩ fluxes) or 0.1 mM (for Mn 2ϩ fluxes) EGTA. MgCl 2 , CaCl 2 , and MnCl 2 were added to obtain a calculated free Mg 2ϩ concentration of 0.5 mM and the indicated concentrations of free Ca 2ϩ and Mn 2ϩ . Free concentrations of Ca 2ϩ and Mn 2ϩ were calculated based on the stability constants for EGTA and ATP given by Fabiato and Fabiato (31) (for Ca 2ϩ ) and by Martell and Smith (32) (for Mn 2ϩ ). Thapsigargin (2 M) was added if inhibition of pumping by the SERCA Ca 2ϩ pump was needed. Efflux was performed in 120 mM KCl, 30 mM imidazole (pH 6.8), and 1 mM EGTA. All experiments on Mn 2ϩ transporting activity or Mn 2ϩ effects on Ca 2ϩ uptake activity were performed with Chelex 100 (Bio-Rad, Eke, Belgium)-treated solutions. To test the sensitivity of the phosphoprotein to hydroxylamine, the samples were additionally incubated for 20 min at room temperature in 0.2 M acetic acid-NaOH with or without 0.2 M hydroxylamine. The samples were then dissolved in a modified SDS loading buffer and subjected to SDS-gel electrophoresis in acid gels as described by Sarkadi et al. (33). The gels were dried between gel drying sheets (Promega) and exposed to screens for quantification of the radioactive bands on a Storm840 scanner in combination with the ImageQuant TM software (Molecular Dynamics, Sunnyvale, CA).
Immunocytochemistry-Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature, washed in phosphate-buffered saline, and permeabilized in 0.2% Triton X-100. Primary and secondary antibodies were diluted in phosphate-buffered saline containing 3% bovine serum albumin at the indicated dilutions.

RESULTS
Gene Structure of C. elegans PMR1 and Alternative Transcript Processing-The C. elegans genome is predicted to contain a single PMR1 orthologue on chromosome I, which is annotated ZK256.1 by the C. elegans sequencing consortium (34). The genomic sequence can be found on the two overlapping cosmids CECC4 (accession number Z81490) and CEZK256 (accession number Z82088). Two EST clones from Yuji Kohara's database, yk218a11 and yk334d5, representing cDNA clones of the C. elegans PMR1 gene, were completely sequenced. The deduced C. elegans PMR1 protein would contain 901 amino acids instead of the 922 predicted in the database annotation (protein identification number CAB04015.1). This is because a sequence of 63 bp is wrongly assigned by the Genefinder program to the putative exon 8 (303 bp). We found this sequence to be absent from both cDNA clones (see Fig. 1A) and hence the actual protein should correspondingly be 21 amino acids shorter. Furthermore, it was clear from clone yk334d5 that the 5Ј UTR of C. elegans PMR1 is at least 257 bp long and contains, besides exon 1, two extra exons, comprising only untranslated sequences and designated exon Ϫ1 and exon Ϫ3 for reasons discussed below (Fig. 1B, second line). Exon 1 (399 bp) starts at position Ϫ87 relative to the ATG start codon, which corresponds to nucleotide 23653 on cosmid CECC4. Exon Ϫ1 (105 bp) and exon Ϫ3 (65 bp) correspond, respectively, to nucleotides 23111-23215 and 15957-16021. This implies that exon Ϫ3 is located more than 7 kb upstream from exon Ϫ1. EST clone yk218a11 does obviously not contain the entire 5Ј UTR, as only the last part of exon 1 is represented in this clone.
A comparison of the exon/intron layout of CePMR1 to that of the D. melanogaster (accession number AC014929) and human orthologues (gene ATP2C1, Refs. 22 and 23) shows that the human gene contains the most elaborate exon/intron layout, D. melanogaster the simplest, whereas C. elegans takes an intermediate position (Fig. 1B). Apparently C. elegans and to a larger degree D. melanogaster have lost most of the introns during evolution. The worm and human have in total 5 conserved exon boundaries, the worm and the fly only one. The localization of the exon 5/6 boundary in CePMR1 corresponds to the exon 1/2 boundary in D. melanogaster and to the exon 18/19 junction in the human gene. There are four extra conserved exon/intron positions between C. elegans and the human ATP2C1 gene (asterisk in Fig. 1B) while there is no other exon/intron junction at homologous positions between the C. elegans and D. melanogaster PMR1 genes. One extra exon boundary is conserved between exons 2 and 3 in D. melanogaster and between exons 21 and 22 in the human ATP2C1 gene. Among the exon borders of the PMR1 family, only one (exon 1/2 in CePMR1, exon 5/6 in the human ATP2C1) is conserved in the mammalian SERCA genes (exon 4/5 in human ATP2A1-3). Compared with the mammalian PMCA family, none of the exon/intron borders is conserved.
Because in C. elegans the majority of the mRNAs are transspliced, we have investigated whether this is also the case for transcripts of CePMR1 in the worm. 5Ј Rapid amplification of cDNA ends experiments were performed on whole worm RNA. PCR reactions with primer pairs SL1/CePMR1Rev and SL2/ CePMR1Rev resulted in the amplification of an 824-and 818-bp fragment, respectively. The 818-bp PCR product corresponded to a cDNA in which splice leader SL2 was transspliced to exon Ϫ3 and exon Ϫ3 was in turn spliced to exon Ϫ1, i.e. an exon layout as found in EST clone yk334d5. By sequencing the SL1/CePMR1Rev fragment of 824 bp, we found that SL1 was spliced to a novel exon (Ϫ2) in the 5Ј UTR of the gene, which corresponds to nucleotides 22950 -23020 on cosmid CECC4 (Fig. 1B, upper line). Thus it appears that the C. elegans transcripts are both alternatively spliced and transspliced at their 5Ј end. Both types of splicing are coupled: a mRNA transcript containing exon Ϫ1 and exon Ϫ2 is transspliced to SL1, a mRNA containing exon Ϫ1 and exon Ϫ3 is trans-spliced to SL2.
We also performed 3Ј rapid amplification of cDNA ends with primer pair CePMR1For/468 to check for the possibility of 3Ј alternative processing at the 3Ј end of the gene's transcript. PCR amplification gave a product of about 600 bp. By subcloning and sequencing of several individual clones, it became clear that the gel band actually consisted of a mixture of three products with small differences in length and corresponding to three polyadenylation isoforms. The isoforms are the result of the use of three polyadenylation sites (pA 1 , pA 2 , and pA 3 ) in the 3Ј UTR of the gene (Fig. 1B). Both yk218a11 and yk334d5 apparently used pA 2 . However, no indication was found for 3Ј alternative splicing, neither in our 3Ј rapid amplification of cDNA ends experiments nor by database searching.
In summary, the C. elegans PMR1 gene consists of 12 exons (Ϫ3 to 9) of moderate length and spans a region of more than 19 kb of genomic DNA. The transcripts are both alternatively and trans-spliced at their 5Ј end. At their 3Ј end, however, no alternative splicing could be documented but alternative polyadenylation occurs.
The C. elegans PMR1 Protein- Fig. 2 shows the predicted amino acid sequence of C. elegans PMR1 and its major domains together with an alignment with the corresponding sequences of three distant species, Homo sapiens, D. melanogaster, and S. cerevisiae. In the sequences of all species 10 hydrophobic segments can be identified, which like in the SERCA Ca 2ϩ pumps, presumably form the transmembrane domain. The highest degree of sequence similarity occurs around the phos-phorylation site, in regions demonstrated in SERCA to contribute to the ATP-binding site or to form structurally important loops, and in transmembrane segments M4, M6, and M8, which have been documented in SERCA to form the binding sites for Ca 2ϩ (35,36). The overall amino acid sequence is 37% identical to rat SERCA2a. The percentage identity with the PMR1 sequences of human, D. melanogaster, and S. cerevisiae is, respectively, 59, 57, and 49%. The amino acids that form the binding site for one of the transported Ca 2ϩ ions in SERCA, more specifically the site II, are conserved in the PMR1 homologues in all species. The amino acids belonging to site I are not conserved in the PMR1 protein. In S. cerevisiae, Gln 783 has been demonstrated to define the Mn 2ϩ selectivity of the PMR1 ion pump (21). Also this residue is conserved in all species.
The N-terminal region upstream of the first transmembrane domain of C. elegans PMR1 has the same length as in the human PMR1 sequence reported by Sudbrak et al. (23), whereas the human sequence predicted by Hu et al. (22) is 16 residues longer. The length of the C-terminal part is more similar to the product of the human splice variant ATP2C1a than to the shorter ATP2C1b described by Hu et al. (22). As mentioned above, there is no evidence for the generation of C-terminal PMR1 protein variants in C. elegans. The C-terminal sequence does not contain an eleventh hydrophobic region as in the SERCA2b splice variant. The EF hand-like domain near the N terminus of the S. cerevisiae sequence has been shown to play a role in modulating ion transport (37). Because its primary structure is poorly conserved, it remains to be demonstrated whether a similar function occurs in other species.
Characterization and Functional Analysis of C. elegans PMR1 Protein-The Celpmrloop antibody, raised against the large cytosolic loop between transmembrane segments 4 and 5 of PMR1 of C. elegans, clearly demonstrated the expression of the protein in COS-1 cells transfected with the corresponding cDNA, both by Western blot analysis and immunocytochemistry (Fig. 3). On Western blots, the immunoreactive band migrated slightly below the predicted theoretical M r value of 98,505. A strong immunoreaction was also seen on blots of fragmented membranes prepared from whole worms. Hence, it is clear that our antibody is able to recognize the PMR1 protein both in C. elegans and after its ectopic expression in COS-1 cells. The vertebrate PMR1 homologue found in untransfected COS-1 cells appears not to react with the antiserum as shown by the controls of untransfected COS-1 cells. Furthermore, immunocytochemistry of PMR1-overexpressing and control cells reveals the correct targeting of PMR1 to the Golgi compartment of the COS-1 cells (Fig. 3B). In conclusion, by using our polyclonal anti-PMR1 antiserum it became clear that PMR1 is expressed in the worms, that it can be overexpressed in COS-1 cells and that it contains all the information needed to target the PMR1 protein to the Golgi membranes.
Because the fraction of Golgi-derived membranes in microsomes of COS-1 cells is relatively small compared with that of ER, we could not rely on conventional techniques used to measure Ca 2ϩ transport, like those for the ER-based SERCA transport ATPases (25). Instead we took advantage of the 45 Ca 2ϩflux system utilizing detergent-permeabilized cells (30).
In a first series of experiments we tested PMR1 of C. elegans for its ability to transport Ca 2ϩ . Control COS-1 cells, cells overexpressing rabbit SERCA1a (as a positive control for Ca 2ϩ pumping), and cells overexpressing PMR1 were permeabilized with saponin in a medium mimicking a cytosolic composition and loaded with 45 Ca 2ϩ for 45 min in the presence of NaN 3 to prevent mitochondrial Ca 2ϩ uptake. Ca 2ϩ transport via SERCA-type Ca 2ϩ -ATPases was determined by comparing the uptake in the presence and absence of 2 M thapsigargin. The loading of the cells was followed by an efflux for 20 min in a Ca 2ϩ -free medium. IP 3 (10 M) was administered after 10 min of efflux. Fig. 4A shows that control and SERCA1a-transfected cells exhibit a Ca 2ϩ uptake, which is blocked by 2 M thapsigargin. In control COS-1 cells and in PMR1-expressing cells, the difference in Ca 2ϩ content with thapsigargin and without thapsigargin represents the Ca 2ϩ pump activity of the endogenous SERCA2b. In SERCA1a-transfected cells, it represents that of endogenous SERCA2b plus overexpressed SERCA1a. In contrast, COS-1 cells overexpressing PMR1 show an additional Ca 2ϩ pump activity even in the presence of thapsigargin. This suggests that PMR1 is a Ca 2ϩ -transporting protein residing in the Golgi and that it is insensitive to thapsigargin. Fig. 4A also shows that Golgi membranes contain IP 3 receptors, since 10 M IP 3 induced a more rapid decrease in the store Ca 2ϩ content. Fig. 4B shows the time course of Ca 2ϩ uptake by PMR1expressing cells. Transfected cells were loaded with 45 Ca 2ϩ for different time intervals in the presence of thapsigargin to block Ca 2ϩ uptake by endogenous SERCA2b. Ca 2ϩ pump activity reaches almost a plateau after 20 min of Ca 2ϩ loading. Subsequent experiments were performed after 10 min of loading with Ca 2ϩ . Fig. 4C shows the Ca 2ϩ dependence of Ca 2ϩ uptake by PMR1. The Ca 2ϩ concentration needed for half-maximal activation was 0.25 M Ca 2ϩ .
In a second series of experiments we explored the possibility of PMR1 of C. elegans to function as a Mn 2ϩ pump, since previous reports based on the activation of ATP hydrolysis by Mn 2ϩ and on the inhibition of Ca 2ϩ transport by Mn 2ϩ suggested that yeast PMR1 could act as a Mn 2ϩ pump. Control cells and PMR1-expressing cells were loaded with radioactive Mn 2ϩ for 10 min in the presence of thapsigargin. The efflux was followed for 10 min (Fig. 5A). Fig. 5, A and C, provide direct evidence that PMR1 can indeed act as a Mn 2ϩ -transporting protein. PMR1-overexpressing cells show an enhanced uptake of Mn 2ϩ . The accumulated Mn 2ϩ was released by the ionophore A23187 (10 M), demonstrating that the Mn 2ϩ has been transported into a membrane-delineated compartment. However, the addition of IP 3 did not have a significant effect on the rate of efflux. The Mn 2ϩ uptake was inhibited by Ca 2ϩ (Fig. 5C), and conversely the Ca 2ϩ uptake by PMR1 was inhibited by Mn 2ϩ (Fig. 5B). It is clear from Fig. 5B that at higher Ca 2ϩ concentrations more Mn 2ϩ was needed to inhibit the transport of Ca 2ϩ . Half-maximal inhibition was observed at 1, 0.5, and 0.25 M Mn 2ϩ for loading at, respectively, 1.0, 0.32, and 0.1 M Ca 2ϩ .
Formation of the Phosphoenzyme Intermediate-A determining characteristic of all P-type ion-transport ATPases is the transient transfer of the ␥-phosphate of ATP to the protein, forming a covalent bond with the carboxyl group of a conserved aspartic acid residue in the large cytosolic domain. The radioactively labeled phosphoprotein can be preserved during SDS-gel electrophoresis by quenching the reaction in acid and maintaining acid conditions throughout electrophoresis. Fig. 6 shows the radioactively labeled phosphointermediate in an SDS gel of microsomes from COS-1 cells overexpressing PMR1. The labeling was completely removed by treatment with hydroxylamine, demonstrating that the phosphate was bound to a carboxyl and not to a hydroxyl group (data not shown). As for the protein detected on Western blots, the phosphoprotein migrated slightly faster relative to the markers than expected from the predicted M r of 98,505. The anomalous migration is probably due to the gel system because a similar shift is also observed for the SERCA2a Ca 2ϩ -transport ATPase, which has a predicted M r of 109,720 (Fig. 6A). The phosphointermediate formation was stimulated by Ca 2ϩ and Mn 2ϩ . The maximum levels were observed below 1 M for both Ca 2ϩ and Mn 2ϩ and these maximum levels were not significantly different (data not shown). There remains a small residual amount of phosphoprotein also in the presence of EGTA without added Ca 2ϩ or Mn 2ϩ . At present we do not have a straightforward explanation for this background labeling. Possibly, a small fraction of the transport-protein molecules in the COS-1 cell membranes is able to reach a conformational state that allows phosphorylation without occupation of the transport sites. The Ca 2ϩ -or Mn 2ϩ -dependent phosphoprotein formation was strongly inhibited by 50 M La 3ϩ (Fig. 6B). The phosphorylation experiments thus confirm the high affinity of the C. elegans PMR1 transporter for both Ca 2ϩ and Mn 2ϩ . DISCUSSION The analysis of the C. elegans genome has resulted in the previous identification of several members of the superfamily of P-type Ca 2ϩ -transport ATPases. A single gene encoding a member of the SERCA-type subfamily is found on chromosome III (cosmid K9D11) and three genes (mca1-3) encoding members of the PMCA subfamily reside on chromosome IV (38). In this work, another gene (designated CePMR1) is identified encoding a P-type Ca 2ϩ -transport ATPase that is located on chromosome I. The conservation of some intron positions, the overall sequence similarity of the encoded protein, and the conservation of major domains and critical motifs of the pri- mary sequence unequivocally place it in the PMR1 subfamily of P-type Ca 2ϩ -transport ATPases. Besides the CePMR1 genomic sequence, we have in this work also characterized its transcripts and protein product.
With respect to the number of exons, the CePMR1 gene takes an intermediate position between the human and D. melanogaster orthologues. Only some of the exon/intron borders are conserved between these different species. All splice sites follow the GT . . . AG rule. Surprisingly, with the exception of the intron between exon 2 and 3, all introns in this gene are relatively long considering the fact that most of the introns in C. elegans genes have a length of only about 50 nucleotides (39). A particularly long intron (ϳ7 kb) is that between exons Ϫ3 and Ϫ2, both located in the 5Ј UTR. Interestingly, we observed the possibility of alternative splicing in the 5Ј UTR, which was coupled to trans-splicing to SL1 or SL2. The meaning of such an alternative trans-splicing which only affects the 5Ј UTR remains unknown. It should be noted that for the human PMR1 orthologue (gene ATP2C1), 5Ј-end alternative splicing has also been suggested but here it affects the open reading frame (23), whereas in C. elegans it does not result in the formation of distinct protein isoforms. At the 3Ј end of the CePMR1 gene three polyadenylation sites were predicted and also experimentally detected by PCR analysis. However, there was neither any predicted nor any experimental indication for alternative splicing. Both for the corresponding human gene and for the rat gene alternative splicing and different protein tails have been suggested. However, the alternative splice sites in human and rat appear not to be conserved.
At the protein level, the major domains described in other P-type transport ATPases can be recognized in C. elegans PMR1. Also sequence motifs demonstrated to be critical for function in other P-type transport ATPases are conserved in the C. elegans sequence (Fig. 2). On the basis of these comparisons, it can be firmly concluded that the coding sequence identified in the C. elegans genome and whose protein product has been investigated in this study is a member of the family of PMR1 ion-transport ATPases. This conclusion is further substantiated by the transport and phosphorylation studies on the protein expressed in COS-1 cells.
Functional data on the PMR1 transporter are up till now available only for the yeast S. cerevisae (21,37,40). In the present work the first characterization of the ion transporting activity of an animal PMR1 enzyme is presented. The C. elegans PMR1 protein overexpressed in COS-1 cells showed a predominantly Golgi-like distribution as shown by immunocytochemistry. ATP-dependent uptake of 45 Ca and 54 Mn was demonstrated in cells permeabilized with saponin. Cells overexpressing PMR1 accumulated more Ca 2ϩ and Mn 2ϩ than control cells, and this additional uptake was not diminished by the SERCA-specific inhibitor thapsigargin, demonstrating that PMR1 is not only able to transport Ca 2ϩ but also Mn 2ϩ . Part of this thapsigargin-insensitive Ca 2ϩ pool was released by IP 3 , which is compatible with existing evidence that the Golgi apparatus is an IP 3 -sensitive Ca 2ϩ store (6). The rate of 45 Ca 2ϩ uptake as determined in our experimental system was halfmaximal at 0.25 M Ca 2ϩ , which is slightly higher than the value determined from the ATPase activity of the purified PMR1 protein of S. cerevisiae (21). The 45 Ca 2ϩ uptake was progressively inhibited by increasing concentrations of Mn 2ϩ . Half-maximal inhibition occurred at a Mn 2ϩ concentration that is about the same as the half-maximal Ca 2ϩ concentration for stimulating the uptake, indicating that the affinity of the C. elegans PMR1 transport ATPase for Ca 2ϩ and Mn 2ϩ is approximately the same. A similar competition between Ca 2ϩ and Mn 2ϩ has been observed for the ATPase activity of S. cerevisiae PMR1, but the yeast showed a somewhat higher affinity for Mn 2ϩ than for Ca 2ϩ (40). Conversely, the uptake of Mn 2ϩ was inhibited by Ca 2ϩ , further confirming the competition between Ca 2ϩ and Mn 2ϩ for binding to the transport sites.
The phosphorylation experiments further validated the conclusion that the high-affinity transport sites can be activated either by Ca 2ϩ or Mn 2ϩ . Phosphoprotein formation was maximal at submicromolar concentrations of Ca 2ϩ or Mn 2ϩ , confirming the high affinity for both cations derived from the competition between Ca 2ϩ and Mn 2ϩ for transport. The phosphoprotein formation was strongly inhibited by La 3ϩ . This effect is in the same direction but more pronounced than that observed with the SERCA-type Ca 2ϩ pumps, whereas the phosphoprotein level of PMCA-type Ca 2ϩ -transport ATPases is increased by La 3ϩ (41).
In conclusion, we have identified and characterized the C. elegans homologue of the PMR1 ion-transport ATPase previously characterized only in yeast. We have shown that the PMR1 protein overexpressed in COS-1 cells shows a predominantly Golgi-like distribution and that its ion transport activity can be measured following permeabilization of the plasma membrane. These experiments directly demonstrate that C. elegans PMR1 is able to accumulate Ca 2ϩ and Mn 2ϩ with high affinity into the Golgi membranes. Part of the accumulated Ca 2ϩ can be released by IP 3 , confirming the observation of Pinton et al. (6) that the Golgi apparatus is an IP 3 -sensitive Ca 2ϩ store.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase.

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.

Nitric-oxide synthase (NOS) 1 catalyzes the NADPH-dependent conversion of L-arginine to nitric oxide and L-citrulline (for reviews, see Refs. 1 and 2). Three isoforms of NOS have been identified in mammals; neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, and their activities are Ca 2ϩ /calmodulin-dependent, whereas inducible NOS (iNOS) is independent of intracellular Ca 2ϩ concentration. Although the three isoforms share 50 -60% sequence identity, they differ in size, intracellular location, and regulation. nNOS has a molecular mass of 165 kDa, is located in neurons in the brain and at neuromuscular junctions, and is involved in neurotransmission. eNOS has a molecular mass of 133 kDa, is present in vascular endothelial cells, and is involved in vascular homeostasis. iNOS has a molecular mass of 130 kDa, is located in macrophages, and is expressed only in response to endotoxin or inflammatory cytokines. A distinguishing feature of NOS is that the N-terminal half of the enzyme is the hemebinding domain (or oxygenase domain), similar to the cytochrome P450 enzyme family, and also contains pterin-and arginine-binding sites. The C-terminal half is the flavin-binding or reductase domain containing FAD-, FMN-, and NADPHbinding sites, as found in NADPH-cytochrome P450 oxidoreductase (CYPOR) (3). At the junction between these two domains is a calmodulin (CaM)-binding region. Due to the reversible binding of CaM, the constitutive isoforms (nNOS and eNOS) display Ca 2ϩ dependence in their enzymatic activities (4,5), whereas iNOS has tightly bound CaM, almost independent of the Ca 2ϩ concentration (6). The reductase domain can be further divided into two subdomains, each of which contains one of the two flavins. The FMN-containing subdomain is situated at the N terminus, and its amino acid sequence is similar to that of flavodoxin, whereas the C-terminal portion contains FAD-and NADPH-binding sites and is homologous to ferredoxin NADP ϩ reductase (FNR). From the sequence alignments, Salerno et al. (7) showed additional sequences in the middle of the FMN subdomain of the constitutive NOS isoforms that were not present in iNOS, CYPOR, or flavodoxin. It was proposed that this additional sequence of about 40 residues acts as an autoinhibitory element in the constitutive NOS isoforms that is released upon binding of Ca 2ϩ /CaM, as seen in other Ca 2ϩ /CaM-activated proteins, such as myosin light chain kinase. Deletion of this insert in nNOS (8) and eNOS (9,10) results in enzymes that require significantly lower Ca 2ϩ concentrations for optimal activity. In addition, removal of this insert greatly enhances the maximal activity of eNOS, which is the least active of the three isozymes. Another significant difference between NOS and CYPOR is that the NOS isozymes contain an additional 21-42-amino acid tail in the C terminus, not present in CYPOR and FNR, which shares about 42% sequence homology with the FAD/NADPHbinding domains of CYPOR and NOS. Roman et al. (11,12) have shown that the C-terminal tails of all three NOS isozymes control electron flow within and through the reductase domains and modulate the effect of Ca 2ϩ /CaM, although the mode and extent of the modulation appear to be different in each isozyme.
The three-dimensional structures of the oxygenase domains of iNOS (13,14), eNOS (14,15), and nNOS 2 have been determined. All three structures are very similar to one another but have no similarities to known cytochrome P450 structures. The remarkable similarities among the structures of different NOS isoforms indicate that isoform-specific regulation of NO production is not obvious from the crystal structures of the heme domains alone. Furthermore, it emphasizes the role of the reductase domain in regulation of NO production in each of the three isoforms. To further investigate the isoform-specific modulation of NO production and its mechanism, knowledge of the structure of the reductase domain of all three isoforms is imperative. Although the structure of NADPH-cytochrome P450 oxidoreductase, a close homolog of the NOS reductase domain, has been determined (16), structural determination of the NOS reductase domain has not been successful, presumably due to proteolysis during the crystallization process. To circumvent this problem, we initiated the x-ray structure determination of its most stable domain from proteolysis, which includes the FAD-and NADP(H)-binding subdomains. Here, we report the crystal structure of the FAD/NADPH-binding domain of rat nNOS (referred to hereafter as nNOS-FAD/NBD (nNOS FADand NADP ϩ -binding domain that includes residues 963-1407)) obtained by limited trypsin treatment of the Escherichia coliexpressed holo protein.

EXPERIMENTAL PROCEDURES
Enzyme Purification-Rat nNOS was overexpressed in E. coli and purified as described previously (17). Briefly, the procedure involved centrifugation of crude cell extract and affinity chromatography on a 2Ј,5Ј-ADP-Sepharose 4B column (Amersham Pharmacia Biotech). The protein was eluted with a buffer solution containing 2 mM 2Ј-AMP and 0.5 M NaCl. The heme-containing fractions were pooled and concentrated, and L-arginine and tetrahydrobiopterin were added to final concentrations of 1 mM and 200 M, respectively. The enzyme was further purified on a gel filtration column (Superose 6HR 10/30; Amersham Pharmacia Biotech), and peak fractions corresponding to nNOS dimers were collected. The purified holo nNOS was treated with immobilized trypsin (Sigma), and the cleaved nNOS protein was further purified on a 2Ј,5Ј-ADP-Sepharose column. The flow-through fraction containing the heme domain (red) was saved for other uses. The material bound to the column (yellow; FAD/NADPH-binding domain) was eluted with 2Ј-AMP and further purified on a gel filtration chromatography column. The purified protein, nNOS-FAD/NBD, was subjected to N-terminal sequencing.
Biochemical Characterization of nNOS-FAD/NBD-The ability of nNOS-FAD/NBD to reduce cytochrome c and ferricyanide was studied as described previously (18,19), with the exception that all assays were performed in the presence of 0.25 M NaCl. The protein concentration was estimated based on the flavin content using an extinction coefficient of 21,600 M Ϫ1 . Because of the effect of ionic strength on the reduction rates, parallel determinations were carried out in the presence and absence of 250 mM NaCl. Recombinant rat NADPH-CYPOR was prepared as described previously (20). N-terminal sequencing (Beckman Porton LF 3000) and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis (Voyager DEPRO; Applied Biosystems) were performed at the Protein Nucleic Acid Facility at the Medical College of Wisconsin.
Crystallization and Data Collection-The purified nNOS-FAD/NBD protein was concentrated to 10 mg/ml in 30 mM potassium phosphate buffer, pH 7.6, containing 0.3 mM NADP ϩ . Crystals were grown by the hanging drop vapor diffusion method at 4°C by mixing 1.5 l of the protein solution with 1.2 l of a precipitant solution (9% polyethylene glycol 4000, 0.1 M Hepes, pH 7.5, and 0.4 M sodium formate) and equilibrating against the precipitant solution. Small thin plate crystals appeared within 24 h and grew to their maximum size over 2 weeks. Crystals of size 0.2 ϫ 0.3 ϫ 0.01 mm were soaked in a cryoprotectant solution (20% glycerol in 11% polyethylene glycol 4000, 0.1 M Hepes, pH 7.5, and 0.3 M sodium formate) for about 3 min before flash-freezing under a liquid nitrogen stream. The diffraction data were collected at 100K on a charge-coupled device detector (CCD Quantum 4) at beamline BioCARS 14BM-C at the Advanced Photon Source at Argonne National Laboratory, Argonne, IL. The crystal-to-detector distance was 160 mm, and the oscillation angle for the data collection was 0.5°. A total of 360 frames were recorded covering 180°oscillation range, and the exposure time was 8 s/frame. The crystal diffracted to 1.9 Å resolution, and the data were processed with the DPS-MOSFLM (21) and CCP4 (22) programs. The crystal belongs to the space group P2 1 2 1 2 1 with the unit cell dimensions a ϭ 59.07 Å, b ϭ 64.37 Å, and c ϭ 159.43 Å. Assuming one molecule/asymmetric unit, the calculated Matthews coefficient is 3.13 Å 3 /Da, corresponding to ϳ60.7% solvent content. Data collection statistics are summarized in Table I. For biochemical characterization of the crystalline protein, several crystals from the crystallization dips were collected, washed with the reservoir solution, and dissolved in a buffer solution. This crystalline protein was analyzed for N-terminal sequencing and also subjected to MALDI-TOF mass spectrometric analysis to determine the C-terminal sequence.
Structure Determination-Initial phases were obtained by the molecular replacement method with the AmoRe program package (23), using the structure of the corresponding domain of rat CYPOR (16) (Protein Data Bank code, 1AMO) as the search model. The initial rotation/translation search gave an R value of 52.4% and a correlation coefficient of 18.4 for the data between 10.0 and 4.0 Å resolution. The subsequent rigid body refinement lowered the R value to 51.4% and increased the correlation coefficient to 31.4. This model was subsequently refined using the CNS program package (24). Each cycle of refinement included maximum likelihood simulated annealing, conjugate-gradient minimization, and individual B-factor refinement. Bulk solvent correction was applied throughout the refinement process. All data within the resolution range of 30 to 1.9 Å were used for the refinement, excluding 8% of reflections randomly extracted for cross- where F o and F c are the observed and calculated structure factors, respectively. b R free is equal to R crst for a randomly selected 8% subset of reflections not used in the refinement. validation (R free ) purposes. After each round of refinement, the model was rebuilt using the TURBO-FRODO program (25). Simulated annealing omit maps were used to locate the peptide regions that have different conformations from the CYPOR structure. After six rounds of refinement and rebuilding, refinement converged with an R free of 20.9% and an R work of 18.8% for all data lying between the 32.0 and 1.9 Å resolution range. The final model contains 435 amino acid residues, a FAD and a NADP ϩ , 462 water molecules, and 4 glycerol and 4 formate molecules. A Ramachandran plot showed that 89.7% of all residues lie in the most favored regions, with 9.3% in additionally allowed regions and 1.1% in generously allowed regions. The final refinement statistics are given in Table II.

RESULTS AND DISCUSSION
Protein Characterization-Purified nNOS-FAD/NBD protein is bright yellow in color and exhibits a spectrum typical of flavoproteins (Fig. 1). The protein was at least 95% pure based on SDS-polyacrylamide gel electrophoresis analysis, with an estimated molecular mass of ϳ50 kDa (Fig. 1, inset). The Nterminal sequence is Ser-Trp-Lys-Arg-Asn . . . , suggesting that trypsin cleavage occurred at the Arg 962 -Ser 963 bond of the rat neuronal NOS holoenzyme. The crystalline protein has the same N-terminal sequence, indicating that no further proteolysis had occurred, at least at the N terminus, during the crystallization process. The MALDI-TOF mass spectrometry result shows that the crystalline protein has a mass of 50,838 atomic mass units, indicating that the C terminus of the protein is Arg 1407 . This suggests that the C-terminal 22 amino acids have been cleaved, presumably by proteolysis during the limited trypsin treatment or possibly (but less likely) during the crystallization process.
Flavoprotein-mediated Reductase Activity of nNOS-FAD/ NBD-The ability of nNOS-FAD/NBD to reduce electron acceptors of different sizes was studied and compared with that of rat CYPOR (Table III). The ability of nNOS-FAD/NBD to reduce cytochrome c is very low, but it is reproducible. To address this residual activity, diphenyleneiodonium, an inhibitor of NADPH-and flavin-mediated electron transport, was employed (26); a 100 -200 times molar excess of inhibitor over the enzyme results in only 20 -25% inhibition of cytochrome c reduction (data not shown). On the other hand, the reaction was inhibited by 80% in the presence of both superoxide dismutase and catalase, suggesting that the low rate of cytochrome c reduction observed with nNOS-FAD/NBD is primarily superoxide-mediated. The ferricyanide activity of nNOS-FAD/NBD is about 20% higher than that of CYPOR, 33% lower than the reductase domain of nNOS, about the same as the full-length nNOS protein in the absence of CaM (data not shown), and about 57% and 67% of those for the nNOS full-length enzyme and the reductase domain of nNOS, respectively, in the presence of CaM. These results indicate that nNOS-FAD/NBD retains its ability to accept reducing equivalents from NADPH and to transfer them to FAD and to ferricyanide.
The Overall Structure-The final model of nNOS-FAD/NBD contains 435 residues starting from Ser 963 to Val 1397 , and the last 10 C-terminal residues are disordered, suggesting the flexible nature of this terminus. The overall polypeptide fold is, as expected, essentially the same as that of the corresponding domain of CYPOR. The structure is composed of three subdomains, as in the case of the CYPOR structure (16), and these subdomains, from N to C termini (Fig. 2, from bottom to top), are as follows: (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADP(H)-binding domain. The connecting domain is not contiguous in the linear amino acid sequence, as it is in the CYPOR structure. The root mean square (rms) difference for the C ␣ atoms in the FAD-binding and NADP ϩbinding domains is 1.3 Å between the structures of the two enzymes, excluding the insertion/deletion regions and the entire connecting domain. Fig. 3 shows a ribbon diagram of a superposition of the two structures. The NADP ϩ -binding domain is shown on the right, the FAD-binding domain is shown in the middle, and the connecting domain is shown on the left. The connecting domain shows the most difference from the structure of the corresponding domain of CYPOR (rms difference, 3.9 Å), reflecting the lower sequence homology in this part of the molecule. The structure of the two nucleotide-binding domains, the FAD-and NADP(H)-binding domains, is also very similar to that of FNR (27), phthalate dioxygenase reductase (28), and cytochrome b 5 reductase (29). A structure-based amino acid sequence alignment of nNOS-FAD/NBD, CYPOR, and FNR together with the sequences of the corresponding domains of eNOS and iNOS is shown in Fig. 4.
FAD-binding Site-As in the FAD domains of CYPOR, FNR,  1. Visible absorption spectrum and SDS-polyacrylamide gel electrophoresis analysis of nNOS-FAD/NBD (inset). The spectrum is that of a typical flavoprotein. The inset indicates the purity of the protein (Ͼ90%) and a molecular mass of ϳ50 kDa. and cytochrome b 5 reductase and the phthalate dioxygenase reductase FMN domain, the nNOS FAD domain has the ␤-barrel structural motif and is composed of six ␤-strands. The N terminus of the only ␣-helix in this subdomain (␣1 in Fig. 4) caps one end of the ␤-barrel, and its N terminus is pointing to the pyrophosphate group of FAD, stabilizing the negative charge with its helix dipole (Fig. 2). The FAD has an extended conformation, and its pyrophosphate group also makes a hydrogen bond to the positively charged Arg 1173 (figure not shown). The isoalloxazine ring of FAD is bound at the interface between the FAD and NADPH domains (Fig. 2). Just as in the structure of CYPOR, the pyrimidine side of the isoalloxazine ring has extensive hydrogen bonding interactions with the polypeptide and with two tightly bound water molecules (Fig.   5). The O2 atom of the isoalloxazine ring makes hydrogen bonds with the main chain atoms of Ala 1193 (amide nitrogen) and Thr 1191 (carbonyl oxygen), the latter of which, in turn, makes a hydrogen bond with the N3 atom of FAD. The N1 atom of the FAD ring is involved in this hydrogen-bonding network via a water molecule (W1 in Fig. 5) to the carbonyl oxygen of Ala 1193 , the O2 atom, and the 4Ј-OH group of the ribityl chain. The O4 atom of the isoalloxazine ring is also linked to Thr 1191 via another water molecule (W2). This intricate network of hydrogen bonds must play an important role in FAD binding/ orientation and is likely to be crucial in stabilizing semiquinone/hydroquinone forms of FAD during electron transfer from NADPH to FAD and from FAD to FMN. The residues implicated in the hydride transfer reaction in CYPOR (30) are also structurally conserved in nNOS-FAD/NBD; the hydroxyl group of Ser 1176 (corresponding to Ser 457 of CYPOR) makes hydrogen bonds to Asp 1393 (Asp 675 of CYPOR) and the O4 atom of the flavin ring, is located near the N5 atom of FAD (3.7 Å), and is coplanar with the isoalloxazine ring. The carboxylate of Asp 1393 makes a hydrogen bond with His 1032 (2.8 Å; His 319 in CYPOR) and is near Cys 1349 (3.7Å; Cys 630 in CYPOR). On the other hand, the xylene ring side of FAD is exposed to the surface of the molecule, strongly suggesting that in the holoenzyme, the FMN domain would interact through this side and receive the reducing equivalents from FAD. The FAD ring is sandwiched between two aromatic rings, Phe 1395 at its re-face and Tyr 1175 at the si-face, serving as stacking residues.
The NADP ϩ -binding Domain-Similar to the NADP ϩ domains of CYPOR and FNR and the NAD ϩ domains of cytochrome b 5 reductase and phthalate dioxygenase reductase, the nNOS NADP ϩ domain has the ␣␤␣ structural motif, with a five-stranded ␤-sheet sandwiched between ␣-helices. Unlike the CPYOR and FNR structures, where the ribose-nicotinamide ring of NADP ϩ is disordered, the current structure clearly shows that the nicotinamide moiety of NADP ϩ is bound at the surface of the molecule, and the cofactor has a bent conformation in which the nicotinamide and adenine rings point to the same side. Fig. 6A shows the vicinity of the NADP ϩ -binding site. A distinct feature in this binding site is the presence of three arginine residues interacting with the phosphate groups of the cofactor: Arg 1010 makes a salt bridge with the pyrophosphate group, Arg 1314 balances charges with the 2Ј-phosphate of the AMP moiety, and Arg 1284 (lying in the middle of and parallel to the two other guanidinium side chains) balances the negative charges of the phosphates. The interaction between Arg 1314 and the 2Ј-phosphate group of the AMP moiety of the cofactor is most likely a major discriminating factor between NADPH and NADH as a substrate. All three Arg residues are conserved in CYPOR and in the other two NOS isozymes. Although the ribose-nicotinamide moiety is well-ordered in this structure, it is located at the surface of the molecule and does not involve any strong interactions with the polypeptide chain (Fig. 6A), implying that its binding is not as tight as that of the 2Ј-ADP portion of the cofactor. As shown in the mutant studies with both CYPOR (30) and FNR (31), it is most likely that during hydride transfer, the phenyl ring of Phe 1395 (Trp 677 of CYPOR), which covers the re-face of the isoalloxazine ring of FAD, swings away to make room for the nicotinamide moiety to bind at the re-face of the isoalloxazine ring. Fig. 6B shows a comparison of the nNOS-FAD/NBD-NADP ϩ structure with that found in the mutant CYPOR structures. Minor dihedral angle changes at the pyrophosphate linkage would transform the bent conformation of NADP ϩ to the extended conformation in which the nicotinamide ring can replace the phenyl side chain of Phe 1395 and directly interact with the flavin ring for the hydride transfer reaction. The exact mechanism and energetics of this conformational change are not clear at present.
The Connecting Domain and the FMN Domain Binding Sites-Approximately 150 residues of nNOS-FAD/NBD constitute the connecting domain, consisting mainly of ␣-helices (Figs. 2 and 4). This domain is unique among CPYOR family members that have both FAD-and FMN-binding sites in a single polypeptide chain, which include NOS, CYPOR, bacterial P450-BM3, methionine synthase reductase (32), and the ␣ subunit of sulfite reductase (33). The connecting domain is not  present in FNR, cytochrome b 5 reductase, and phthalate dioxygenase reductase. Accordingly, its folding is also unique. However, it has the least sequence similarity with CYPOR within the entire nNOS-FAD/NBD sequence, and this is reflected in the structural alignment. The rms difference between the C ␣ atoms in the two structures is 3.9 Å, as compared with the corresponding value of 1.3 Å for the other two domains (FAD/ NADPH domains). The N-terminal residue Ser 963 corresponds to Ser 241 of CYPOR in the end of the hinge region that connects the FMN-and FAD-binding domains. It is reasonable to assume that the nNOS reductase domain also has a flexible hinge connecting the two flavin domains and is therefore susceptible to proteolysis. As shown in Fig. 4, the linear amino acid sequence of this domain is not contiguous; instead, it makes an excursion (three ␤-strands consisting of about 50 residues) at about 30 residues from the N terminus into the FAD domain. This results in an intertwined tight binding and close interface between the connecting domain and the rest of the nNOS-FAD/ NBD molecule, in contrast to the interface with the FMN domain.
A closer examination of the connecting domains of nNOS-FAD/NBD and CYPOR reveal small but significant structural differences between the two molecules. The two most significant differences lie in the first 27 residues at the N terminus and the loop between strands ␤6 and ␤7 (Glu 1077 to Asp 1091 , Fig. 4), both of which are located at the surface that might interact with the FMN domain. The N-terminal 27 residues (Ser 963 -Lys 989 ), 12 residues shorter than the corresponding region in CYPOR (Ser 241 -Lys 279 ), share no obvious sequence homology to CYPOR, and, accordingly, their secondary structures also differ. In nNOS, residues Asp 979 -His 988 form a regular ␣-helix, whereas the corresponding part of CYPOR adopts a loop structure. On the other hand, region Glu 1077 -Asp 1091 in nNOS contains 8 extra residues compared with the CYPOR molecule. These residues form a long ␤-stranded finger, extrud-ing out of the molecule into the solvent region, whereas the corresponding region in CYPOR makes a short loop that is tightly bound to the rest of the molecule. At the tip of the finger lies a hydrophobic loop with residues 1072 Ala-Lys-Gly-Val-Ile, suggesting that this loop is most likely buried in the presence of the FMN domain and possibly CaM. Interestingly, a structure-based sequence alignment (Fig. 4) shows that this loop is also short in iNOS as in CYPOR. The distance between the tip of this loop and the insertion point of the putative autoinhibitory element described by Salerno et al. (7) is about 27 Å. Considering the size of the autoinhibitory loop (ϳ45 residues), it is possible that these two loops interact with each other in response to Ca 2ϩ /CaM binding. It should be noted that the autoinhibitory domain, inserted in the middle of the FMN domain, is present only in the constitutive NOSs and absent in iNOS and CYPOR, both of which do not respond to Ca 2ϩ /CaM concentration.
Superposition of the nNOS-FAD/NBD with the CYPOR structure (Fig. 3) also reveals the binding site for the FMN domain, the location of the autoinhibitory loop insertion, and their relative positions to the rest of the structure. The putative FMN-domain binding site lies mainly on the connecting domain. Fig. 7 shows the electrostatic surface potentials of the interface of nNOS-FAD/NBD that might interact with the FMN domain, along with the corresponding surface of CYPOR and the complementary surface of its FMN domain. In the case of CYPOR, the interaction between the two domains (the FMN and FAD/NBD domains) is highly electrostatic, with the former being negative, and the latter being positive (Fig. 7, D and C,  respectively). The corresponding surface of nNOS-FAD/NBD is less charged (Fig. 7B) The cofactor is shown in red, with its nitrogen atoms in blue. NADP ϩ is bound at the surface of the enzyme molecule and adopts a bent conformation. The 2Ј-phosphate makes salt bridges with Arg 1314 and hydrogen bonds with Tyr 1322 and Ser 1313 ; the pyrophosphate group makes salt bridges with Arg 1010 and a hydrogen bond with Thr 1251 . The plane of Arg 1284 is parallel to and sandwiched between the planes of Arg 1010 and Arg 1314 . Whereas the phospho-AMP moiety makes extensive interactions with the polypeptide, the ribose-nicotinamide portion is loosely bound, suggesting conformational flexibility of the cofactor during catalysis. B, superposition of the NADP ϩ structure observed in nNOS-FAD/NBD (dark blue) with that of a productive mode found in the mutant CYPOR (red) (30). The 2Ј-AMP moiety common to both conformations is shown in green. In order for the hydride transfer reaction to occur, the nicotinamide group must replace the phenyl side chain of Phe 1395 , which occupies the re-face of the FAD ring. Minor adjustments of dihedral angles at the pyrophosphate linkage transform a resting conformation (blue) to an active (or productive) conformation (red). based sequence comparison of the two proteins and two other NOS isozymes (Fig. 4), only two residues, Glu 1067 (corresponding to Asp 352 of CYPOR) and Arg 1229 (Arg 514 of CYPOR), on the nNOS-FAD/NBD surface are conserved. The other three residues are either replaced with neutral residues or absent due to a shorter loop in all three NOS proteins. Another distinctive feature of all NOS isozymes, differentiating them from the CYPOR protein, is their longer C termini, extended by 33 residues in nNOS, 42 residues in eNOS, and 21 residues in iNOS. It has been shown that these C-terminal tails control electron flow into, between, and out of the two flavins in all three isoforms and affect modulation by calmodulin in the constitutive NOSs (11,12). The facts that the C-terminal 22 residues are missing from the crystalline protein and that the adjacent 10 residues are disordered in the crystal structure suggest that the C-terminal 32 residues form a separate structural subdomain and probably lie near the interface between the two flavin domains. Thus, it is likely that this subdomain modulates the interflavin distance and, in response to Ca 2ϩ / CaM concentration, regulates the electron flow between the two flavins and out of FMN by controlling the access to FMN of various electron transfer partners, including cytochrome c and the oxidase domain.
Conclusions-Our current structural analysis confirms the structural similarity between CYPOR and the nNOS reductase domain (and presumably the other NOS isozymes). It also confirms that the structure of the FAD/NADPH domain is very similar to those of the members of the FNR superfamily, including FNR, cytochrome b 5 reductase, and phthalate dioxygenase reductase. A closer comparison between the structures of nNOS-FAD/NBD and CYPOR, however, reveals not only important similarities but also some significant differences between the two enzymes (Figs. 3 and 4). The residues shown to be catalytically important for the hydride transfer mechanism in CYPOR (Ser 457 , Cys 630 , Asp 675 , and possibly His 319 ) are all structurally conserved in nNOS-FAD/NBD (Ser 1176 , Cys 1349 , Asp 1393 , and His 1032 , respectively) (Fig. 4). In addition, the tightly bound water molecules involved in the hydrogen bonding network at the pyrimidine side of the isoalloxazine ring of FAD and an aromatic residue stacking at the re-face of the flavin ring (Phe 1395 in nNOS and Trp 677 in CYPOR) are also conserved. This constellation of residues in the vicinity of the isoalloxazine ring of FAD strongly suggests that the hydride transfer mechanism of nNOS is very similar to that of CYPOR suggested by Hubbard et al. (30). Also, this hydrogen bonding network is most likely responsible for stabilization of the semiquinone/hydroquinone form of FAD during catalysis, and a water molecule (W2 in Fig. 4) might act as a proton donor/ acceptor during hydride transfer. Ser 1176 is in close proximity to the N5 atom of the flavin ring, and the arrangement of Ser 1176 , Asp 1393 , and Cys 1349 (or His 1032 ) resembles the catalytic triad of the serine protease family, suggesting a proton relay during catalysis. Furthermore, the mode of NADP ϩ binding is also similar in the two enzymes; the 2Ј-ADP moiety is tightly bound to the enzyme, yet the nicotinamide side of the cofactor is either disordered (in CYPOR) or loosely bound (in nNOS) at the surface of the molecule. Recently, Hubbard et al. (30) have shown with CYPOR mutants in which the aromatic residue (Trp 677 ) was either deleted or mutated to glycine that the nicotinamide moiety of NADP ϩ binds at the re-face of the FAD ring in a manner suitable for hydride transfer from the nicotinamide ring to FAD, and they suggested that a conformational change occurs during catalysis involving Trp 677 and the pyridine nucleotide. Modeling of NADPH in a productive conformation similar to the one observed in the mutant CYPOR structure (Ref. 30; Fig. 5B) suggests that Ser 1176 and Asp 1393 are involved in interactions with the nicotinamide ring, thereby orienting and stabilizing the binding of the pyridine nucleotide for the hydride transfer reaction. Site-specific mutagenesis studies are in progress to confirm the role of these residues in the catalytic reaction. Although the FMN domain is not included in the nNOS-FAD/NBD structure, the current structure reveals the putative binding site for the FMN domain and the relative locations of various elements of the reductase domain that control/modulate different catalytic steps involved in cytochrome c reduction and NO production. The exact binding sites of the C-terminal and autoinhibitory domains and their interactions with the rest of the reductase domain and Ca 2ϩ / CaM must await the crystal structure determination of the entire reductase module, including the calmodulin-binding region.