Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11514-11518, December 1995 Biochemistry

Inducible : Identification of amino acid residues essential for dimerization and binding of HEARN J. CHO*t, EMIL MARTIN*t, QIAO-WEN XIE*, SHIGERU SASSAt, AND CARL NATHAN* *Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College and the tRockefeller University, New York, NY 10021 Communicated by Seymour J. Klebanoff University of Washington, Seattle, WA, September 5, 1995 (received for review July 17, 1995)

ABSTRACT Nitric oxide (NOSs) require tet- low-spin state (17). In the present study, we have identified rahydrobiopterin (BH4) for dimerization and NO production. residues critical for the binding of BH4 within one of the Mutation analysis of mouse inducible NOS (iNOS; NOS2) longest regions of conservation among reported NOS se- identified Gly-450 and Ala-453 as critical for NO production, quences (18-20). dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of MATERIAL AND METHODS , , and NADPH militated against major dis- tortions affecting the NH2-terminal portion, midzone, or Mutagenesis and Transfection. Mouse iNOS expression COOH terminus of the inactive mutants. Direct involvement plasmid piNOSL8 (10) was subjected to PCR mutagenesis to of residues 450 and 453 in the binding of BH4 is supported by derive A4531 and C451A variants, and site-specific mutagen- the striking homology of residues 448-480 to a region exten- esis was used to derive A447I, G450A, P452A, and P461A. sively shared by the three BH4-utilizing aromatic amino acid Mutations were confirmed by DNA sequencing. Human epi- hydroxylases and is consistent with the conservation of these thelial 293 cells (American Type Culture Collection) were residues among all 10 reported NOS sequences, including transiently transfected by using the calcium phosphate method. mammalian NOSs 1, 2, and 3, as well as avian and insect Twenty-eight to 36 h later, iNOS activity was measured in cell NOSs. Altered binding of BH4 and/or L- may explain lysates as described (10) and iNOS protein expression was how the addition of a single methyl group to the side chain of monitored by SDS/PAGE (7.5% gel) and immunoblot analysis residue 459 or the addition of three methylenes to residue 453 with rabbit IgG against pure wild-type mouse iNOS (21). The can each abolish an enzymatic activity that reflects the same IgG was used for all immunoblots and immunoprecipi- concerted function of 1143 other residues. tations described below unless otherwise stated. Blots were developed with goat anti-rabbit IgG conjugated with horse- radish peroxidase as detected by enhanced chemiluminescence The radical nitric oxide (NO) has emerged as an important (10). To produce stable transfectants, 293 cells were co- signaling and cytotoxic molecule in metazoan physiology transfected with a mutant iNOS construct and the pcDNA3 (1-4). NO is synthesized from L-Arg, oxygen, and NADPH by vector (Invitrogen) bearing the neomycin-resistance . variably regulated isoforms of NO synthase (NOS). Products Cells growing in G418 (GIBCO; 600 ,ug/ml) were cloned by of the three known mammalian NOS (4) include two limiting dilution and screened for iNOS production by immu- (NOS1 and NOS3) that are constitutively expressed and noblot analysis. Native iNOS was induced in RAW 264.7 activated by binding calmodulin in response to elevated Ca2+ activated by interferon y and bacterial lipopoly- and one [inducible NOS (iNOS); NOS2] that is activated saccharide as described (11) and was used interchangeably transcriptionally and binds calmodulin without an elevation of with recombinant iNOS from piNOSL8 to serve as the wild- Ca2+ (5). These complex attach at least six molecules type control. besides calmodulin (3, 6): L-Arg, heme, tetrahydrobiopterin Characterization of Mutant iNOSs. Cell lysate (100 ,ul) (BH4), FMN, FAD, and NADPH. For only two of these from transient transfectants was subjected to size-exclusion cofactors or cosubstrates have residues important for binding chromatography in buffer A (40 mM Bis-Tris propane, pH been identified through mutational analysis. These are Cys- 7.2/50 mM NaCl/1 mM L-Arg/3 mM dithiothreitol/2 ,uM 184, which controls the binding of heme in human NOS3 (7) BH4/2 p.M FAD) on TSK (North Bend, WA) G3000SW or and corresponds to Cys-415, which controls the binding of Superdex 200 columns (Pharmacia). Fractions were analyzed heme in rat NOS1 (8, 9), and residues 1121-1144 in mouse by SDS/PAGE and immunoblot analysis. To detect heme, iNOS, which contribute to the binding of NADPH (10). iNOS was immunoprecipitated from transient transfectant The for BH4 is of particular interest for two lysates and the protein A-Sepharose-bound complexes were reasons. (i) BH4 is essential for formation of stable NOS boiled in 2 M oxalic acid. This reaction generates from heme dimers in purified preparations (11, 12) and in cells (T. a stoichiometric amount of protoporphyrin IX, which gives Billiar, personal communication), and dimerization is obliga- a characteristic two-banded fluorescence emission spectrum tory for generation of NO (11, 12). (ii) The BH4 binding site with peaks at 606 and 662 nm (excitation, 400 nm) (22). To is likely to share, abut, or allosterically alter the heme- detect BH4, iNOS was partially purified from lysates of 293 containing catalytic site, since L-Arg and BH4 each increase cells stably transfected with constitutively expressed G450A the affinity for the other in NOS1 (13), L-Arg affects the Soret and A4531 mutant iNOS genes by affinity chromatography peak and CO binding properties of the heme in NOS1 and on 2', 5'-ADP Sepharose (see below) (11) in the absence of NOS2 (8, 9, 14,15), BH4 protects NOSl's heme from NO (16), exogenous BR4 and a 100-,lI sample was treated with 5 p.l of and BH4 deficiency converts much of the heme in NOS1 and 0.1 M HCI and 5 p.l of 0.1 M KI/12 at room temperature for NOS2 from a pentacoordinate high-spin to a hexacoordinate 1 h in the dark. Ascorbic acid (10 p.l, 0.1 M) was added, and

The publication costs of this article were defrayed in part by page charge Abbreviations: NO, nitric oxide; NOS, NO synthase; iNOS, inducible payment. This article must therefore be hereby marked "advertisement" in NOS; DHFR, dihydrofolate reductase; BH4, tetrahydrobiopterin. accordance with 18 U.S.C. §1734 solely to indicate this fact. tH.J.C. and E.M. contributed equally to this work. 11514 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11515 the precipitate was removed by centrifugation. The super- A natant was subjected to C18 reverse-phase HPLC (Waters A 600E), eluting pterins were detected by fluorescence as active mutants I A A described (23), and peak areas were compared to those of '. 7 t tt t 481 standards. Binding of calmodulin was assessed by immuno- iNOS aRgGCPADW WI VPPvSGS TPVFHQEM nYvLsP precipitating iNOS from lysates of transient transfectants (1.5 mg of protein), SDS/PAGE (15% gel), and immunoblot inactive mutants A I analysis with anti-calmodulin monoclonal antibody (0.1 ,tg/ml) (5). Binding of NADPH was assessed by affinity chromatography B (10). Lysates of transient transfectants (2-4 mg of protein) were applied to a 2', 5'-ADP Sepharose column equilibrated with < << c) < buffer A. Nonspecifically bound proteins were washed out with CD " r ,.J ° ,s, e buffer A containing 0.5 M NaCl and the column was reequili- 4 Lo f brated with buffer A containing 0.12 M NaCl. Proteins able to m X bind to NADPH were eluted with buffer A supplemented with 8 mM NADPH and examined by SDS/PAGE and immunoblot analysis. C

RESULTS 0 E Activity of iNOS Mutants. Constructs expressing mutant or 0) wild-type iNOS (Fig. 1A) were transiently expressed in 293 CJ) cells to determine NOS activity. When activity was consid- ~>, E ered in light of the level of iNOS protein expression (Fig. ¢r a 1B), Ala substitution for the conserved residues Cys-451, .0 C Pro-452, or Pro-461 allowed substantial enzyme as ._ z activity, a) Q- + did substitution of Ile for the nonconserved Ala-447 (Fig. Ul), 1C). P461A was relatively more active in intact cells (as reflected by accumulation of in their medium) than in z cell lysates (Fig. 1C), suggesting that this mutant might be less stable than wild type upon dilution. In contrast, the mutants G450A and A4531 accumulated no nitrite in their - I'l 0 cm: culture medium and their lysates displayed no NOS activity '- LO LO LO uL ( 0 (Fig. 1C), despite levels of expression comparable to that of < cD QI2 0- <: a active mutants and wild type (Fig. 1B). Inactivity of G450A and A4531 persisted at reduced temperature (30°C) (data not FIG. 1. Mutations of conserved and nonconserved residues of shown). When wild type and G450A in a 1:5 molar ratio or iNOS. (A) Active mutants of mouse iNOS are indicated above and wild type and A453I constructs in molar ratios of 1:1, 1:2, or inactive mutants are indicated below the sequence of iNOS residues 1:5 were cotransfected, no inhibition of iNOS activity was 447-481. Capital letters denote residues completely conserved in evident. Thus the G450A and A4531 mutations can be eight sequences reported for three isoforms of NOS in mouse, rat, considered recessive lethals. cow, and human (18) as well as in NOSs from chicken (19) and Drosophila (20). Nonconserved residues are in lowercase type, Homodimerization. Since only dimeric iNOS produces NO including Leu indicated by at positions 458 and 475, not to be (11, 12), we asked whether G450A and A453I might be confused with Ile (I). (B) Immunoblot estimation of iNOS protein enzymatically inactive because of a defect in dimerization. expression in transient transfectants. (C) Enzyme activity of lysates Gel filtration chromatography of native iNOS in lysates of from 293 cells transiently transfected with wild-type and mutant activated RAW 264.7 cells (Fig. 2A) or recombinant wild- iNOSs for the number of experiments shown above the bars. Open type iNOS in lysates of transient transfectants (Fig. 2B) bars, activity in intact cell monolayers as assessed by accumulation resolved iNOS antigen into two broad peaks centered at of nitrite over 20 h in their conditioned medium; shaded bars, activity in cell lysates expressed as pmol of NO2 plus NO3 per mg - 300 kDa and - 150 kDa, corresponding to homodimers and monomers. As before (11, 12), only proteins in the dimer of protein per min. peak could generate NO (Fig. 2A). In contrast, G450A and determined by immunoblot analysis (Fig. 4B), released no A4531 were eluted almost exclusively as monomers (Fig. 2B). detectable The predominantly monomeric distribution of A4531 was BH4 (Fig. 4A). confirmed by nondenaturing gel electrophoresis (data not Calmodulin and NADPH Binding. To probe the structural shown). integrity of regions of iNOS distant from the inactivating Heme Binding. Since monomers of wild-type iNOS require mutations, we tested the binding of calmodulin and NADPH. heme, BH4, and L-Arg to dimerize (12), we next asked whether Calmodulin is thought to attach to residues 503-532, which are the dimerization defect of G450A and A4531 might be sec- presumed to adopt a basic amphipathic helical configuration in ondary to inability to bind one or more of the latter. As shown the intact protein (21, 24, 25). Immunoprecipitates of the in Fig. 3, similar amounts of protoporphyrin IX were extracted inactive mutants G450A and A4531, the active mutant C451A, from the immunoprecipitates of wild-type iNOS, inactive and wild-type iNOS contained similar amounts of calmodulin mutants, and an active mutant. Thus, all bound heme. More- (Fig. SA). over, Cys-451 can be excluded as a candidate for providing the The residues confirmed to be required for NADPH binding heme's thiolate ligand. support a structural model in which the COOH-terminal BH4 Binding. In two experiments, one of which is illustrated portion of iNOS shares the five-stranded (3-sheet configuration in Fig. 4A, 0.78 and 7.8 pmol of native wild-type iNOS released adopted by spinach ferredoxin:NADP+ reductase (10). A4531 0.92 and 5.2 pmol of BH4, respectively, for an average recovery (Fig. SB) and G450A (data not shown) bound NADPH of 0.92 mol of BR4 per mol of iNOS monomer. In contrast, normally, suggesting that the COOH-terminal portion of iNOS comparable amounts of affinity-purified G450A and A4531, as is not perturbed. 11516 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 92 (1995)

A 30

A-c '% C1)(S 20 C 0Q (0) cz 1A4531 E

0 Z0 (a C451A Fraction: .E w Western Blot:

G450A 300 kDa 150 kDa 560 680 5460 680 B Emission, nm wt iNOS FIG. 3. Binding of heme. Lysates of 293 cells transiently transfected -* **- with pcDNAI vector alone or with wild-type or mutant iNOS were G450A immunoprecipitated with anti-iNOS IgG and their extracts were assayed for protoporphyrin IX fluorescence. Immunoblots confirmed A4531 that the amount of iNOS protein in the immunoprecipitates from A4531 and G450A was no less than for wild type (data not shown).

300 kDa 150 kDa the DHFR module could mean that they contribute indi- rectly but importantly to the module's conformation or, FIG. 2. Interchain binding. (A) Lysate from activated RAW 264.7 alternatively, that the DHFR module does not actually bind macrophages as a positive control was chromatographed on a TSK BH4. In any event, with 501 amino acids, the biopterin- G3000SW gel-filtration column. Fractions eluted between 13 and 17 sensing peptide of NOS1 is not much smaller than the ml were assayed for activity and examined by immunoblot analysis with anti-iNOS IgG. Two broad peaks of iNOS antigen were centered at proteolytically generated hemimers of NOS1 (residues -300 kDa and -150 kDa, representing dimeric and monomeric iNOS, 1-714) and NOS2 (residues 1-502) shown to bind BH4 (29, respectively (11, 12). Only the dimer peak was associated with enzyme 30). activity. (Inset) Calibration of the column with molecular mass stan- Mutation analysis, the approach used here, has the potential dards: bovine thyroglobulin (670 kDa), bovine y-globulin (158 kDa), to identify individual residues critical to BH4 binding while ovalbumin (44 kDa), and equine myoglobin (17 kDa). Elution volume introducing minimal structural perturbation. Pending solution is expressed as ml. (B) Anti-iNOS immunoblots of gel-filtration of the tertiary structure of NOS, the possibility that the fractions of lysates of 293 cells transiently transfected with wild-type mutations G450A and A4531 affected BH4 binding through and mutant iNOSs. Wild-type iNOS was eluted in both dimeric and monomeric peaks, whereas A4531 and G450A were eluted primarily as distant structural perturbation is best evaluated in the context monomers. The samples were chromatographed on a Superdex 200 of the number, nature, and impact of the mutations tested and column. the extent of homology to regions in other proteins sharing the postulated function. The following considerations militate DISCUSSION strongly against the possibility that A453I and G450A abro- gated iNOS's BH4 binding, dimerization, and NO generation Identification of BH4 as a of iNOS (26, 27) permitted the through gross distortion of its tertiary structure. purification of iNOS (11, 26), which led in turn to the (i) The substitutions were highly conservative. (ii) There demonstration of bound FAD (11, 28), FMN (11, 28), and was no adverse impact of five other substitutions lying within calmodulin (5). To our knowledge, no mutational analysis has 1-8 residues of the two sites identified as critical. These identified residues involved in the binding of any of these four included four mutations introduced experimentally and one cofactors. We have focused on the binding of BH4 as a step natural variant representing Ala -- Ile at position 456 when toward characterizing its mechanism of action and as an human and bovine NOS3 are compared to mouse iNOS (18). approach toward identifying the binding site for L-Arg, with Likewise, the same Ala -> Ile mutation that devastated iNOS which BH4 appears to interact. at position 453 was inconsequential when introduced at Limited proteolysis of both NOS1 (29) and NOS2 (30) position 447. Even neighboring mutations that might disrupt released an NH2-terminal hemimer containing BH4 and disulfide bonds (C451A) or helicity (P452A and P461A) were heme. Two strategies have been brought to bear to narrow relatively benign. down the BH4 binding site. Nishimura et al. (31) noted that (iii) The normal binding of heme, calmodulin, and NADPH rat NOS1 amino acid residues 558-721 (corresponding to excluded major distortions affecting the NH2-terminal portion, mouse iNOS residues 333-491) can be modeled on the midzone, or COOH terminus of iNOS. Retention of heme is tertiary structure of a BH4-utilizing enzyme, chicken dihy- particularly noteworthy, since BH4 protects the heme from NO drofolate reductase (DHFR). Consistent with this, a recom- (16) and other ligands at the sixth coordination site (17), binant fragment containing residues 220-720 of NOS1 was implying that the heme-binding and BH4-binding sites of NOS capable of BH4-enhanced binding of Nw-nitro-L-arginine orient their respective ligands toward a common cavity. (31), although direct binding of BH4 was not demonstrated. The direct involvement of residues 450 and 453 in the However, BH4 failed to enhance the binding of Nw-nitro-L- binding of BH4 is supported by the striking homology of arginine to a recombinant fragment of NOS1 corresponding residues 448-480 to a region extensively shared by each of the to the postulated DHFR module itself, that is, residues BH4-utilizing aromatic amino acid hydroxylases (Fig. 6) and 558-721 (31). The requirement for residues not included in confirmed by mutational and serologic analysis to be critical to Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11517 A

!A' 1 2 3 4 5 A 1.2 -. RH. qtAnHri rci 11 anti-iNOS 0.6 IgG _ mAb 0.0 .4h___ anti-CaM

...... _.

RAW ~ ~ 1 B 0.14 RAW +C 1 2 3 4 5 6 7 8 0.120

0.10 .. .? ~ flow through 500 mM NaCI 0.20 A4531 9 10 11 12 13 14 0.16 E

0.12 - " 8 mM NADPH FIG. 5. Binding of calmodulin and NADPH. (A) Lysates of 293 0.16 mock cells transiently transfected with pcDNAI vector alone or with wild- type or mutant iNOS were immunoprecipitated with anti-iNOS IgG 0.14 and the samples were divided for immunoblot analysis with either anti-NOS IgG or anti-calmodulin (anti-CaM) monoclonal antibodies 0.12 (mAb). Lanes: 1, pcDNA I vector transfectant; 2, wild-type iNOS; 3, A4531; 4, G450A; 5, C451A. (B) Affinity purification of A4531 on 2', 2 4 6 8 10 5'-ADP-Sepharose as monitored by anti-iNOS IgG immunoblot. 0.04 _ Lanes: +C, positive control consisting of lysate of 293 cells transiently G450A i transfected with A4531; 1-4, flow-through fractions; 5-8, fractions collected during wash with 500 mM NaCl; 9-14, fractions eluting with 0.00 8 mM NADPH.

-0.04 Binding of L-Arg to NOSI has been modeled kinetically ° 3 6 9 1 2 with the aid of analogs such as N@-nitro-L-arginine (13). With iNOS, the off-rate of such analogs may be much faster Minutes than with NOSI; experimental values are low (E.M., unpub- lished data). Nonetheless, in two experiments in which

< .0o LI Nw-nitro-L-arginine binding by wild-type iNOS was demon- cc < E a:c strable, no binding by A4531 was detectable (E.M., unpub- lished data). Thus it remains an unresolved issue whether the primary impact of mutations A4531 and G450A is on BH4 FIG. 4. Binding of biopterin. (A) Lysate from activated RAW 264.7 binding, L-Arg binding, or intermonomer interaction. A macrophages as a positive control was affinity-purified on 2', 5'-ADP defect in any one of these three functions might interfere Sepharose. As a negative control, the same procedure was applied to with the other two. 293 cells transfected with the pcDNAI vector lacking an iNOS insert. It is remarkable that changes as subtle as the addition of Experimental samples were purified in the same manner from 293 cells a single methyl group to the side chain of residue 450 or the stably transfected with either A4531 or G450A. All four samples were acid-oxidized and their supernatants were subjected to reverse-phase addition of three methylenes to residue 453 can each abolish HPLC on a C1s column. Eluting biopterin was detected by excitation an enzymatic activity that reflects the concerted function of at 350 nm and fluorescent emission at 440 nm as compared to a BH4 1143 other residues. Further mutations, covalent binding of standard eluting at the position indicated by the arrow. The top four analogs of BH4 and L-Arg, and solution of the tertiary parts were run at one flow rate and the bottom panel (G450A) was run structure should refine our understanding of the critical at another flow rate. Standards and lysates from RAW cells and mock contributions of the side chains normally displayed at these transfectants were also run at the latter flow rate but are not shown. (B) Anti-iNOS IgG immunoblot of affinity-purified preparations positions. confirmed that the amount of iNOS protein in the immunoprecipitates from A4531 and G450A was no less than for wild type. iNOS RG - WITWLVP-FVSGST - 1MM YV Phe Hydroxylase RV HC TOY IIRHGSKPMYTPEP IC L HV the ability of Phe hydroxylase to bind BH4 (37, 38). Finally, the Trp Hydroxylase RV LYT PEP TC H extensive conservation of this region among all 10 reported Tyr Hydroxylase C CTQYIVIRHSSDP-TQY IRHASSP-MHSPEP CC L H ML NOS sequences-insect (20), bird (19), and mammalian (18)- supports the premise that the conserved region subserves a DHFR [VLSY-JPGPLDTNMQ-OLARETSMDP- RSRLOKL function as critical as the binding of a cofactor and/or sub- DHPR LGSfRl-VQAFRARNWW-VAEDVIeNE- SAS[qIVK strate. Chen et al. (39) have reported that the C99A mutant of FIG. 6. Region of iNOS from residues 448-480 that is highly human NOS3 (corresponding to mouse iNOS residue 109) lost conserved among NOSs (see Fig. 1) is homologous to a BH4-binding domain that is conserved rat aromatic amino acid catalytic activity, dimerization, and BH4; heme was highly among binding hydroxylases (32-34). Residues shared by both classes of enzymes preserved (7). No further analysis was provided to aid in the (NOSs and aromatic amino acid hydroxylases) are boxed. A lesser distinction between direct and indirect effects of replacement degree of homology exists between the same region of iNOS and of this Cys. sequences from rat DHFR (35) and dihydropteridine reductase (36). 11518 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 92 (1995) We are indebted to Dr. Steven Gross for generous help with assays Conference, eds. Ignarro, L. & Murad, F. (Univ. 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