Identification of a Mouse Short-chain Dehydrogenase/Reductase Gene, Retinol Dehydrogenase-similar

We report a mouse short-chain dehydrogenase/reductase (SDR), retinol dehydrogenase-similar (RDH-S), with intense mRNA expression in liver and kidney. The RDH-S gene localizes to chromosome 10D3 with the SDR subfamily that catalyzes metabolism of retinoids and 3α-hydroxysteroids. RDH-S has no activity with prototypical retinoid/steroid substrates, despite 92% amino acid similarity to mouse RDH1. This afforded the opportunity to analyze for functions of non-catalytic SDR residues. We produced RDH-SΔ3 by mutating RDH-S to remove an “additional” Asn residue relative to RDH1 in its center, to convert three residues into RDH1 residues (L121P, S122N, and Q123E), and to substitute RDH1 sequence G208FKTCVTSSD for RDH-S sequence F208-FLTGMASSA. RDH-SΔ3 catalyzed all-trans-retinol and 5α-androstane-3α,17α-diol (3α-adiol) metabolism 60–70% as efficiently (Vm/Km) as RDH1. Conversely, substituting RDH-S sequence F208FLTGMASSA into RDH1 produced a chimera (viz. C3) that was inactive with all-trans-retinol, but was 4-fold more efficient with 3α-adiol. A single RDH1 mutation in the C3 region (K210L) reduced efficiency for all-trans-retinol by >1250-fold. In contrast, the C3 area mutation C212G enhanced efficiency with all-trans-retinol by ∼2.4-fold. This represents a >6000-fold difference in catalytic efficiency for two enzymes that differ by a single non-catalytic amino acid residue. Another chimera (viz. C5) retained efficiency with all-trans-retinol, but was not saturated and was weakly active with 3α-adiol, stemming from three residue differences (K224Q, K229Q, and A230T). The residues studied contribute to the substrate-binding pocket: molecular modeling indicated that they would affect orientation of substrates with the catalytic residues. These data report a new member of the SDR gene family, provide insight into the function of non-catalytic SDR residues, and illustrate that limited changes in the multifunctional SDR yield major alterations in substrate specificity and/or catalytic efficiency.

The short-chain dehydrogenase/reductase (SDR) 1 gene superfamily encodes ϳ100 bacterial, plant, and animal members related through a limited number of conserved residues that determine structure, provide for cofactor binding, and catalyze dehydrogenation/reduction (1)(2)(3). SDR family members do not always share substantial amino acid identities and have relatively few strictly conserved residues. Animal SDR catalyze intermediary metabolism and activation/inactivation of nuclear receptor ligands such as prostaglandins, retinoids, and steroid hormones. SDR tend to have multifunctional catalytic abilities: they can catalyze reactions with dissimilar substrates and/or can recognize functional groups in different loci of the same substrates. An apparent subgroup of the SDR superfamily consisting of phylogenetically related enzymes catalyzes dehydrogenation of all-trans-retinol, cis-retinols, and androgens or reduction of retinals. Functions of this subgroup could include serving in the visual cycle, generating the endocrine factors all-trans-retinoic acid and 9-cis-retinoic acid, reducing retinal produced by carotenoid metabolism, and/or reactivating 5␣-androstane-3␣,17␣-diol (3␣-adiol) into dihydrotestosterone (4,5).
Of the mouse SDR in the retinoid subfamily, RDH1 has widespread expression and seems to have the highest catalytic efficiency for all-trans-retinol dehydrogenation; mouse 17␤-HSD9 catalyzes all-trans-retinol dehydrogenation about an order of magnitude less efficiently than RDH1, and RNase protection assays reveal 17␤-HSD9 mRNA expression only in liver (6,7). Other mouse SDR such as CRAD1 and CRAD3 catalyze 9-cis-retinol dehydrogenation much more efficiently than alltrans-retinol dehydrogenation and have weak, if any, activity with all-trans-retinol (8,9). CRAD2 has very low efficiency for all-trans-retinol and even lower efficiency for 9-cis-retinol (10). RDH4 catalyzes all-trans-retinol dehydrogenation at least 2 orders of magnitude less efficiently than RDH1 (11,12). The mouse SDR retSDR1, RRD, and PSDR1 function as reductases that convert all-trans-retinal into all-trans-retinol, but do not catalyze dehydrogenation of all-trans-retinol (13)(14)(15)(16). Therefore, although an array of SDR catalyze retinoid metabolism, RDH1 represents the only SDR thus far in the mouse with high catalytic efficiency for all-trans-retinol dehydrogenation and widespread tissue expression, with expression initiating early during embryogenesis.
Here we report cDNA cloning of a novel mouse SDR gene and determination of its mRNA expression pattern and its chromo-somal localization. This SDR, termed retinol dehydrogenasesimilar (RDH-S), shares 92-95% amino acid similarity with mouse RDH1, CRAD1, and CRAD3, but does not have enzyme activity with their prototypical substrates. This afforded the opportunity to investigate the residues that influence substrate specificity and catalytic efficiency for retinoids and androgens. We prepared RDH1/RDH-S chimeras and mutants of RDH-S and RDH1 and determined their catalytic activities with retinoids and steroids. We show that minor alterations activate RDH-S, that one-residue changes in RDH1 have a large impact on efficiency for retinol, and that relatively limited changes alter the specificity and/or catalytic efficiency for either retinol or 3␣-adiol. These data provide new insight into the amino acid residues that contribute to the substrate specificity and catalytic efficiency of SDR and present the possibility that RDH-S may have a regulatory rather than an enzymatic function.
Radiation Hybrid Mapping-A mouse radiation hybrid panel was purchased from Research Genetics. A forward primer (CCCTGGGTAG-GAGGTTCAGTCCCT) and a reverse primer (ACAGGTAAATTCTGT-TCATGGGCTT) were selected from the 3Ј-untranslated sequence of the mRDH-S cDNA for amplification. PCR was done for 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The radiation hybrid PCR experiment was repeated, and the sequence of the amplified 350-bp fragment was confirmed by sequencing. The PCR results were sent for analysis to the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research. 2 Northern Blotting-A probe from the 3Ј-untranslated region of mRDH-S was amplified from the cDNA with the primers used for radiation hybrid mapping. The PCR product was cloned into vector pGEM-T, sequenced, and labeled with [ 32 P]dCTP using the RadPrime DNA labeling system (Invitrogen). The probe was hybridized overnight at 68°C with a mouse multiple-tissue blot (Clontech) according to the manufacturer's protocol. Mouse ␤-actin cDNA was used as a control. Blots were exposed to x-ray film with an intensifying screen at Ϫ70°C for 1 day.
Expression of RDH-CHO-K1 cells (American Type Culture Collection, Manassas, VA) were cultured at 37°C in Ham's F-12 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. Cells were transfected with pcDNA3 constructs (8 g/100-mm plate) with LipofectAMINE, harvested 24 h later, and lysed as described (6). The supernatant obtained from centrifuging the lysates at 800 ϫ g for 10 min was used for enzyme and Western blot analyses. Supernatants from CHO-K1 cells transfected with pcDNA3 or pFLAGCMV5a (Sigma) served as controls. Protein concentrations were determined by the dye-binding method (18).
Enzyme Assays-Protein amounts and reaction times were used in the linear range to obtain initial velocity values. Kinetic analyses were done with two to three replicates per point at 37°C in 0.25 ml of 50 mM Hepes, 150 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 2 mM NAD ϩ (pH 8.0) with 2-4 g of protein for 8 -15 min for all-trans-and 9-cisretinol. Analyses of 3 H-labeled steroids (20,000 dpm/reaction) were done with 1-3 g of protein for 1.5-2 min. Each curve was obtained at least twice. Retinoids were quantified by high-performance liquid chromatography. 3 H-Labeled steroids (40 -101 Ci/mmol) were separated by thin-layer chromatography, detected by autoradiography, and quantified using liquid scintillation counting as described (6 -9). Kinetic data were fit by nonlinear regression analysis with Prism Version 3.0 (GraphPAD Software, Inc).
FLAG-tagged Fusion Proteins-To produce FLAG epitope (DYKD-DDDK)-tagged RDH, expression vectors were used as templates with forward primer 5Ј-CGGAATTCCACCATGTGGCTCTACCTGGTT (containing a Kozak sequence) and reverse primer 5Ј-GGGGTAC-CGAGGGCTTTCTCAGG, which moved the stop codon to place the FLAG sequence in-frame at the C terminus. PCR products were digested with EcoRI and KpnI and cloned into pFLAGCMV5a.
Western Blotting-Ten g of cell lysate protein were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with rabbit anti-mRDH1 antibody (1:5000 (v/v); raised against RDH1 peptide D 217 RLSSNTKMIWDKASSEVK) in phosphate-buffered saline/Tween. For FLAG-tagged proteins, mouse anti-FLAG tag monoclonal antibody M2 (Sigma) was used at a dilution of 1:5000 (v/v) in phosphate-buffered saline/Tween. Blots were incubated with either anti-rabbit or anti-mouse secondary antibodies conjugated with alkaline phosphatase (Promega). Bands were visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Molecular Modeling-mRDH1 was modeled using Swiss-Model Version 36.0002. Details of the modeling procedure have been described, including global alignment, model relaxation for possible steric overlaps of the side chains, and energy minimization (19 -21). The BLASTP2 program found all similarities using pairwise alignment of mRDH1 with sequences of three-dimensional structures from the ExNRL-3D Database. The SIM program selected all templates with amino acid sequence identities above 25% and projected model size larger than 20 residues. This step also detected domains that could be modeled based on unrelated templates. This generated ProModII (Version 3.5) input files. Global alignment was done using the loaded templates. ProModII then generated all models using the ExPDB Database. Energy minimization of all models was done with the Gromos96 program.

RESULTS AND DISCUSSION
cDNA Cloning of RDH-S-We sequenced 14 clones encoding SDR after reverse transcription-PCR of mouse E17 embryo mRNA using primers from conserved regions (F 33 ITGCDSGFG and S 179 K(Y/F)G(I/V/L/F/M)EAFSD) of retinoid/steroid-metabolizing SDR. Eight encoded CRAD2 (10); two encoded 17␤-HSD9 (7); three encoded an orphan SDR (22); and one encoded a novel SDR, similar to RDH1, which we named RDH-S. 5Ј-and 3Ј-RACE generated the full-length coding sequence of mRDH-S. The 3Ј-untranslated region was extended further with two rounds of 3Ј-RACE to generate a cDNA of 1890 bp (Fig. 1). The open reading frame encodes a deduced protein of 318 amino acid residues with high homology to other SDR that catalyze retinoid/steroid metabolism (Table I). mRDH-S differs in only 33 residues from RDH1, many of them conservative substitutions (Fig. 2). Notably, RDH-S has the six peptide motifs characteristic of retinoid/steroid-metabolizing SDR (1). Nineteen of the 23 amino acid residues conserved in ϳ70% of SDR occur within these six motifs. Conserved residues include the cofactor-binding sequences T 35 GX 3 GXG and N 111 NAG in the first and second motifs, the catalytic sequence S 164 X 11 YX 3 K in the fourth and fifth motifs, part of the substrate-binding domain in the sixth motif, and the oligomerization domain in the third motif. An additional amino acid, Asn, occurs in RDH-S between residues 173 and 174 (RDH1 numbering), which potentially could change the secondary/tertiary structure relationship between catalytic residues Ser 164 , Tyr 176 , and Lys 180 .
RDH-S mRNA Expression-Northern blot analysis was done with a 3Ј-untranslated region probe. mRDH-S mRNA was intensely expressed in liver and kidney, but expression was not observed in the six other tissues analyzed (Fig. 3). Four sizes of RDH-S Chromosomal Localization-Radiation hybrid mapping indicated that mRDH-S locates to mouse chromosome 10D3 between markers D10Mit269 and D10Mit271 and near CRAD1 (Rdh6) and CRAD2 (Rdh7). The RDH-S gene maps close to seven other members of the subfamily (Fig. 4).
Lack of mRDH-S Enzyme Activity-mRDH-S, which shares 92% amino acid similarity and 89% identity with mRDH1 ( Fig.  2 and Table I), did not catalyze metabolism of the major substrates recognized by mRDH1. These include all-trans-retinol, 9-cis-retinol, 3␣-adiol, and androsterone (data not shown). The lack of RDH-S activity was surprising because none of the residue differences occurred in obviously crucial sections (see above), and many were conservative substitutions. These limited differences between RDH1 and RDH-S afforded the opportunity to distinguish the residues in SDR that contribute to activity with retinoids and steroids.
Distinct Requirements for Steroid Versus Retinoid Activity-The non-conservative differences provide obvious starting points for evaluating the effects of substituting RDH-S residues into RDH1. The RDH1 mutant A7P behaved enzymatically similar to RDH1; therefore, no further analysis was done with this mutant. Substituting the section of RDH-S from residues 117 to 147 into RDH1 produced chimera C1, with nine total and eight non-conservative residue differences from RDH1 (Figs. 2 and 5). C1 had no detectable activity with all-trans-retinol or 3␣-adiol (Fig. 6, A and B, bars 4). C1 mutant L121P/S122N/ Q123E, i.e. C1/PNE, was made because sequence L 121 SQ in C1 differs substantially from P 121 NE conserved in mRDH1, rat RoDH1-3, and the human ortholog RDH-E. C1/PNE had partial activity with all-trans-retinol and 3␣-adiol (Fig. 6, A and B,  bars 5). Mutating any two of the three C1 residues produced chimeras/mutants without activity (C1/PN, C1/PE, and C1/NE) (Fig. 6, A and B, bars 6 -8). Chimera C2 differs from RDH1 in four residues, with one non-conservative change. C2 had par- FIG. 2. mRDH-S and mRDH1 amino acid sequences. White letters on a black background denote substitutions in RDH-S relative to RDH1: non-conservative differences are shown in boldface italics. The boxes labeled with Roman numerals denote the six peptide motifs conserved in retinoid/steroid-metabolizing SDR. Lines above sequences indicate regions of RDH-S substituted into RDH1 to create chimeras, with the specific chimera indicated by the CX notation. The arrowhead shows insertion of an Asn residue in RDH-S between RDH-1 residues 173 and 174. mRDH1 and mRDH-S tial activity with both substrates (Fig. 6, A and B, bars 9). Chimera C2 with Asn inserted between residues 173 and 174 of RDH1, i.e. C2ϩN, was inactive with both substrates (Fig. 6, A  and B, bars 10). Chimera C3 differs in six residues from mRDH1, with three non-conservative changes. C3 was not active with all-trans-retinol, but catalyzed 3␣-adiol metabolism at a higher rate than RDH1 (Fig. 6, A and B, bars 11). Chimera C4, which includes the RDH-S residues of C3 as well as six additional differences, four of which are non-conservative, was inactive with both all-trans-retinol and 3␣-adiol (Fig. 6, A and  B, bars 12). Consequently, only all-trans-retinol activity requires the specific C3 area residues of RDH1, but 3␣-adiol activity requires the residues in the C-terminal part of C4, i.e. Lys 224 , Lys 229 , and Ala 230 . Chimera C5, which partially over- FIG. 4. mRDH-S chromosomal locus. mRDH-S localizes to mouse chromosome 10D3 near the genes that encode the other members of the SDR subfamily that recognize retinoids and steroids as substrates. CRAD-L refers to a CRAD-like protein without retinoid/steroid activity. CRAD-t refers to a truncated mRNA that does not have a complete fourth exon. The chevrons indicate the directions of the genes. SDR-O, SDR-orphan.

FIG. 5. Schematic of RDH1 and RDH-S chimeras.
The open bars indicate amino acid residues from RDH1; the filled bars indicate residues from RDH-S. The numbers above the bars (other than 1 and 317) indicate the beginning of the inserted sequence followed by the return to the RDH1 or RDH-S template sequence, e.g. in C1, 117 indicates the first residue of the RDH-S section inserted into RDH1, and 148 indicates the return to RDH1 sequence. Therefore, the insertion in this case represents RDH-S residues 117-147. In C1/PNE, 121 indicates the beginning of RDH1 residues 121-123 inserted into the section of RDH-S represented in C1, and 124 indicates the return to RDH-S residues. In C1/PE, 122 indicates Ser 122 of RDH-S inserted between Pro 121 and Glu 123 of RDH1. laps with C4, had activity with both all-trans-retinol and 3␣adiol, but not as high as that of RDH1 (Fig. 6, A and B, bars 13), indicating compensation for the deleterious effects of the residues in the C terminus of C4. Chimera C6, which has the C-terminal 76 residues of RDH-S substituted into RDH1, catalyzed a higher rate of metabolism with all-trans-retinol and a lower rate with 3␣-adiol (Fig. 6, A and B, bars 14). This is intriguing because, with one exception (N249S), the nine residue differences are conservative in the C6 sections of RDH1 and RDH-S.
Activity Requires Three Regions-These results obtained above indicated that only three areas in RDH-S abolished enzyme activity: sequence L 121 SQ, the Asn insertion, and the segment from Phe 208 through Ala 217 . Therefore, RDH-S was mutated to remove the Asn residue; to make the conversions L121P, S122N, and Q123E; and to replace F 208 FLTGMASSA with G 208 FKTCVTSSD of RDH1. This construct, RDH-S⌬3, catalyzed the metabolism of both all-trans-retinol and 3␣-adiol at 60 -70% of the rate of RDH1 (Fig. 6, A and B, bars 15). Each of these three changes was made independently in RDH-S, i.e. chimera C7, deletion of only the "additional" Asn residue; chimera C8, substitution of RDH1 P 121 NE for RDH-S L 121 SQ; and chimera C9, substitution of RDH-S F 208 FLTGMASSA with RDH1 G 208 FKTCVTSSD. In addition, the two remaining combinations of any two changes were made in RDH-S, i.e. chimera C10, RDH-S L 121 SQ/P 121 NE and deletion of the Asn residue; and chimera C11, RDH-S F 208 FLTGMASSA/G 208 FKTCVTSSD and deletion of the Asn residue. All of these chimera were inactive.
Changes Occur in Both k cat and K m -Values of kinetic constants were determined for the most active chimeras and mutants (Table II). Converting RDH-S into RDH-S⌬3 created a catalytically active protein with lower K m values compared with those of RDH1 with both all-trans-retinol and 3␣-adiol, albeit one less efficient than RDH1 because of lower V m values. With the exception of RDH-S⌬3, the mutations affected activity with all-trans-retinol and 3␣-adiol much differently. C1/PNE showed major (ϳ10-fold or greater) increases in K m values for both all-trans-retinol and 3␣-adiol, but maintained an efficient k cat only with all-trans-retinol. C3 had no detectable activity with all-trans-retinol, but had ϳ4-fold higher efficiency with 3␣-adiol compared with RDH1, predominantly because of a 5-fold lower K m value. C5 showed substantial efficiency with all-trans-retinol, but was unsaturated kinetically with 3␣adiol. The activity of C6 with all-trans-retinol was only 20% as efficient as that of RDH1, despite a V m value ϳ4-fold higher, but was not saturated kinetically with 3␣-adiol. These results seem remarkable because most of the residue differences in each construct are conservative relative to RDH1, consistent with a large impact of a very few non-conservative changes.
RDH1 Residues 208 -217 Contribute to Retinol Specificity-C3 was assayed with 9-cis-retinol and androsterone because mRDH1 catalyzes metabolism of both (6). C3 had weak activity with 9-cis-retinol, just as it did with all-trans-retinol, but substantial activity with androsterone, just like with 3␣-adiol, which reinforces the conclusion that retinoid recognition requires the specific C3 residues of RDH1, whereas steroid activity can better tolerate substitution (Fig. 7). The K m values of RDH1 and C3 for NAD ϩ were 30 Ϯ 0.9 and 40 Ϯ 9 M, respectively, and those for NADP ϩ were 3.7 Ϯ 0.5 and 18 Ϯ 4.7 mM, respectively (data not shown). Thus, residues 208 -217 do not seem to contribute to NAD ϩ binding or function, and protein folding seems normal in C3.
RDH1 Single-residue Mutants-We performed site-directed mutagenesis on each non-conservative difference among RDH1 residues 208 -217. Two single mutations had opposite effects on all-trans-retinol dehydrogenase activity: K210L and C212G. Mutant K210L was at least 2500-fold less efficient than RDH1 (Fig. 8). This coincides with strict conservation of an Arg/Lys 210 residue in the SDR with measurable retinol dehydrogenase activity, viz. mRDH1, mouse 17␤-HSD9, human RDH-E, and rRDH1-3. CRAD2, which has weak activity with all-transretinol, with a V m /K m ϳ2-3 orders of magnitude less than that of RDH1, also has a conserved Arg residue at position 210. In contrast, CRAD1 and CRAD3 have barely detectable or no activity with all-trans-retinol and have a Leu residue at position 210, like RDH-S. These data suggest that Arg/Lys 210 is necessary but not sufficient for all-trans-retinol dehydrogenase activity.
The RDH1 mutation C212G had a 2.4-fold increase in efficiency produced by an increase in the V m value, without marked impact on the K m value, and presented the only mutation with increased efficiency for all-trans-retinol (Table III). This difference does not appear to reflect a general principle because CRAD1 and CRAD3 have a Gly residue at position 212, and each has negligible or undetectable activity with all-transretinol. In addition, SDR active with all-trans-retinol, such as rRDH1-3 and mouse 17␤-HSD9, have Asp, Glu, and Asn residues at position 212.
The D217A mutant had increased K m and V m values, but maintained the same overall efficiency as RDH1. rRoDH1, rRoDH3, mouse 17␤-HSD9, and human RDH-E have a Glu residue at position 217, but rRoDH2 has a Val residue, consist- mRDH1 and mRDH-S ent with the tolerance observed here in the D217A mutant. Mutants V213M and T214A had reduced efficiencies stemming from increased K m values, despite a 3-fold increase in the V m value in the case of V213M. Most SDR with measurable retinol dehydrogenase activity have Val 213 (RDH1, rRoDH1-3, mouse/ human RDH4/5, and human RDH-E); 17␤-HSD9 provided the exception, with a Met residue like that of RDH-S, but 17␤-HSD9 showed much lower efficiency than RDH1. Each SDR with measurable retinol dehydrogenase activity has a Thr residue at position 214, including 17␤-HSD9, consistent with the noted negative effect of the T214A mutation. These data and activity comparisons among the retinoid dehydrogenase SDR indicate that loss of all-trans-retinol-metabolizing activity with chimera C3 could have been caused by K210L alone, but probably was exacerbated by V213M and T214A.
Differences in Expression Are Not Responsible for Differences in Activities-Western blotting confirmed the expression of RDH1 and the lack of cross-reactivity of the anti-RDH1 antibody with RDH-S (Fig. 6C, lanes 1 and 3). Because the anti-RDH1 antibody does not recognize RDH-S, C-terminally FLAG-tagged chimeras were made of RDH-S and RDH1. Neither the anti-FLAG nor anti-RDH1 antibody reacted with endogenously expressed CHO-K1 cell proteins (Fig. 6, C and D,  lanes 2). The chimeras/mutants tested, including the FLAGtagged proteins, were expressed to the same extent as wildtype RDH1. The enzyme activity of RDH1-FLAG was similar to that of RDH1, demonstrating that the C-terminal FLAG addition did not affect catalytic properties (data not shown). The single-residue mutants also had expression levels similar to those of RDH1 (Fig. 8, lower panel). These data exclude protein expression differences as a major contributor to changes in V m values.
Molecular Modeling-We generated a molecular model based on three-dimensional structures of soluble SDR using an optimal amino acid sequence alignment. The model relied on x-ray structures of human 17␤-HSD type I complexed with cofactor and/or substrate (39.8% identity; Protein Data Bank codes 1FDS, 1A27, and 1EQUB) (23-25), 20␤-HSD (51.5%; Protein Data Bank code 1HU4) (26), and ␤-ketoacyl-(acyl-car-    9. Molecular model of mRDH1. N t indicates residue 28 at the N terminus, and C t shows the C-terminal residue. Green indicates backbone residues that were not the focus of this work. Black depicts the cofactor-binding residues Gly 36 , Gly 40 , and Gly 42 . Orange depicts the catalytic residues Ser 164 , Tyr 176 , and Lys 180 . Dark blue depicts chimera C1 with the P 121 NE sequence depicted in yellow. Yellow shows Gly 173 and Gly 174 , which are interrupted by an Asn residue in RDH-S, indicated by asn. Magenta depicts chimera C3, with Lys 210 and Cys 212 shown in yellow. Red depicts residues 224 -242 of chimera C5. Cyan depicts residues 243-316, which compose chimera C6 and the latter residues of C5.