The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies.

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169-173. Substitutions of Ile-171 and −172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower V value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.

Cytochrome P450 is a protein superfamily of more than 200 members (1). Every member in the superfamily binds heme molecules at their active site to carry out mixed-function monooxygenation reactions at the end of the electron transport chain (2,3). These proteins are termed cytochromes P450, because the reduced heme in the protein shows characteristic absorption at 450 nm upon binding to carbon monoxide (4).
In contrast to the bacterial P450s, eukaryotic P450s are associated with the membrane of the endoplasmic reticulum (ER) 1 or mitochondria. The topology of P450s relative to the membrane has not been elucidated due to a lack of knowledge about the three-dimensional structure. It is generally believed that the bulk of the P450s have a structure similar to that of the bacterial P450s, with an N-terminal hydrophobic domain integrated into the membrane. This N-terminal hydrophobic domain not only serves as the signal for ER membrane targeting and integration (5,6), it also participates in the determination of the overall structure and stability of P450c21 (7). The rest of the polypeptide probably forms a globular structure located at the cytoplasmic phase of the membrane (8 -11), although a part of the polypeptide might interact with the membrane (8).
Cytochrome P450c21 as a microsomal protein is integrated into the membrane of the smooth ER. It catalyzes the conversion of progesterone and 17-hydroxyprogesterone (17-OHP) into deoxycorticosterone and 11-deoxycortisol, two essential steps of steroid hormone synthesis (3). Its deficiency is the main cause of congenital adrenal hyperplasia, due to decreased cortisol synthesis, leading to virilization and sometimes salt loss. 21-Hydroxylase deficiency is mainly caused by mutations of the CYP21A2 (c21B) gene that encodes P450c21 (12,13). c21B and its neighboring CYP21A1P (c21A) genes are more than 98% identical in sequence, but the c21A gene does not encode an active protein due to multiple mutations throughout the gene (14,15). Because of their proximity, these two genes exchange their sequences frequently through gene conversion events. This gene conversion is the main cause for the mutations found in the c21B gene (16) and can be used as the basis for the detection of known mutations in patients (17).
The loss of enzymatic activity as a result of mutations of the c21B gene is shown by the expression of mutant proteins corresponding to each mutation in various cell types in mammalian (18), vaccinia (19), or yeast vectors (20). Some mutations cause the complete loss of enzymatic activity, resulting in the severe salt-wasting type of the disease. Other mutations have less deleterious effects, resulting in mutant proteins with some residual activities, causing the milder form of the disease (21,22). The availability of the expression systems enables the examination of parameters affecting the structure and function of wild type and mutated cytochromes P450. One common mutation is the Ile-172 to Asn substitution (23,24). This mutation causes 100-fold loss of enzymatic activity resulting in the simple virilizing form of the disease (24). In this report, we investigated the structural perturbation caused by the substitution. Despite the 100-fold loss of enzymatic activity causing the simple virilizing form of the disease, the Ile-172 mutation did not grossly affect the integration of the protein into the endoplasmic reticulum. Instead, the mutant protein became more sensitive to proteinase K digestion. This result indicated that the mutant protein had an altered conformation.

EXPERIMENTAL PROCEDURES
Plasmid Construction-Site-directed mutagenesis of the P450c21 cDNA cloned in M13 using a kit was performed as described previously (20). The mutated cDNA was subcloned into p5Јc21 and pYE8 vectors for in vitro transcription/translation and yeast expression, respectively, as described before (7,25).
Immunofluorescent Detection of Proteins-A stable cell line of Rat-1 expressing either wild type or the I172N form of P450c21 (26) was grown on cover slides to 70 -80% confluence. After fixing with ice-cold acetone:methanol (1:1) for 90 s and blocking with 5% bovine serum albumin in phosphate-buffered saline at room temperature for 30 min, the cells were incubated with anti-P450c21 antisera at 1:500 dilution for normal P450c21, or at 1:50 dilution for the I172N mutant and normal rabbit serum for 1 h, followed by washing in phosphate-buffered saline 5 times. Immunofluorescence was detected under a light or confocal fluorescence microscope after incubation of the slides with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (FabЈ, Catalog No. 12120111, Cappel, Belgium) at 1:500 dilution for 1 h in the dark followed by washes in phosphate-buffered saline 5 times.
Analysis of Yeast Microsomes-Yeast microsomes were isolated from a 6-liter culture at one time in the presence or absence of 1 M 17-OHP by modifying published procedures slightly (27,28). After solubilization of proteins in microsomes with 0.5% sodium cholate and 0.2% Emulgen 913 in buffer A (10 mM Tris-HCl, pH 7.5, 0.65 M sorbitol, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride), the substrate-binding and CO-difference spectra of microsomal proteins (0.5 mg/ml) were recorded. The concentrations of cytochrome P450 and P420 were calculated as described earlier (29,30). 21-Hydroxylase activity assay and kinetic measurements were performed as described before (25).
Measurement of Amounts of P450c21 in Yeast-Yeast cells were labeled with 25 Ci/ml of [ 35 S]Met in SD medium without Trp and Met for 1-2 h or were pulse-labeled with 100 Ci/ml of [ 35 S]Met for 5-10 min (31). The cell lysate was immunoprecipitated by anti-P450c21 and anti-hsp60 antisera as described before (7).
Proteinase K Digestion of P450c21-Yeast microsomes (120 g of protein) containing wild type or mutant P450c21 proteins or in vitro translated P450c21 were digested by various concentrations of proteinase K at room temperature for 30 min. The reaction was stopped by 4 mM phenylmethylsulfonyl fluoride before gel electrophoresis. Proteins in the gel was transferred to membranes which were then soaked in 5% nonfat milk in PBST (0.2% Tween 20 in phosphate-buffered saline) for 1 h at room temperature and washed in PBST. Proteins on the membrane were reacted with anti-P450c21 antibody (18) at 1 to 10,000 dilution at room temperature for 1 h followed by three 5-min washes in PBST. The membrane was then soaked in peroxidase-labeled goat anti-rabbit antibody (1 to 10,000 dilution) at room temperature for 1 h before three 5-min washes in PBST. After draining excess solution, the membrane was overlaid with detection reagents (ECL kit, 5 ml each, Amersham) at room temperature for 1 min before exposure to x-ray film for chemiluminescent detection.

Rationale for Site-directed Mutagenesis of P450c21 at
Residues 169 -173-To understand the functional importance of the domain surrounding the site of the common Ile-172 to Asn mutation of P450c21, sequence alignment of many microsomal P450s was made (Fig. 1). Ile-172 is invariant, implying its functional importance. Site-directed mutagenesis was per-formed substituting Ile-172 with either hydrophobic (Leu, Val) or polar (Gln, His) amino acids. Residue 171 is either Ile or Val. It was replaced by Val as found in some P450s or Asn to study the degree of changes that can be tolerated. Other residues in the vicinity are more variable, either hydrophobic or polar. Conservative changes replacing Cys-169 by Thr, Ser-170 by Thr, and Cys-173 by Ser were made separately to avoid dramatic changes of the protein structure. All mutant proteins were expressed in yeast and the levels of protein production, enzymatic activities, and kinetic properties were assayed individually.
Amount of Mutant Proteins-The steady state levels of wild type and mutant P450c21 protein in the cells were detected by immunoblotting (Fig. 2). Antiserum against yeast hsp60 protein was added at the same time to serve as an internal control for the amount of protein loading in each well (32). Most of the mutant proteins were present at levels similar to that of the wild type P450c21, except the I171N mutant protein, which appeared to be present at a lower amount in many independent experiments. Reduced amounts of proteins in the cell usually indicate that either the synthesis of the protein is decreased or that the newly synthesized protein is very rapidly degraded.
To determine synthetic rates of proteins, mutant proteins were pulse-labeled with [ 35 S]Met for 5 min followed by immunoprecipitation. Fig. 3 showed that most of the mutant proteins were synthesized at normal rate except the I171N mutant. Scanning of protein intensities from four separate experiments showed that the I171N protein was produced at 70 Ϯ 16% of the normal rate.
Function and Kinetic Properties of Mutant Proteins-The enzymatic activities of wild type and mutant proteins toward both substrates, progesterone and 17-OHP, were assayed. As shown in Fig. 4A, most substitutions did not greatly affect the enzymatic activity of P450c21, except the mutations changing the conserved residues at 171 and 172 from Ile to Asn. When replacing Ile-172 with four other amino acids, the resulting enzymatic activities were all less than 5% of wild type (Fig. 4B). The enzymatic activities for both substrates paralleled each other, indicating that there is no preferential loss of utilization of one particular substrate. The extremely low activity of all 172 mutants expressed in yeast was also observed in the proteins expressed in COS-1 cells (18), indicating the requirement for Ile at this conserved site.
The kinetic properties of the mutated and wild type enzymes in the yeast microsomes were determined further (Table I). Some P420 forms, which usually represent denatured proteins with altered interaction with heme (30), were associated especially with the mutant protein during purification after cells were broken. Similar to other P450s like P450cam (33) and P450c11 (34), adding the substrate 17-OHP during purification stabilized P450c21 and increased yield of the P450 form for both wild type and mutated P450c21 (data not shown).
We obtained similar amounts of P450s (about 2 nmol/liter) for most of the mutant forms, except the I171N and I172N mutant proteins. The yield for the I171N mutant (1.26 nmol/ liter) was about 50% of the wild type protein (Table I), consistent with the decreased abundance of the apoprotein in the cell. The yield for the I172N P450 (0.29 nmol/liter) was about 10% of that of the wild type. Since P450 content is a measure of the amount of hemoprotein (4), it indicated that there is less hemoprotein although the I172N apoprotein was produced at the normal amount (Fig. 2).
The K m values of all mutants were similar to that of the wild type protein with a slight elevation of the I171N protein (Table  I). This result indicates similar affinity of the mutant proteins toward the substrate. As the I172N mutation does not drastically affect the ability of the protein to bind substrate, the Ile-172 residue of P450c21 does not seem to be involved directly in substrate binding. The V max value of the I172N mutant protein, however, was decreased to about 1/6 to 1/10 that of the normal protein when equal amounts of the P450 were compared. This together with about 10% yield of the I172N P450 results in overall low enzymatic activity.
The I172N Mutant Protein Integrates into the Membrane of ER Normally-The cause of the reduced overall enzymatic activity created by the I172N mutation was investigated further. Amino acids 167-185 of P450b (CYP2B1) could serve as a stop transfer signal and membrane anchoring domain under a certain experimental condition, implying the ability of this motif to traverse the membrane (6). There was also the notion that the Ile-172 to Asn mutation might affect the ability of P450c21 to localize in the ER (19). We therefore tested whether the I172N protein is impaired in ER targeting. Immunofluorescence detection, either under the conventional light microscope or a confocal microscope, showed that the wild type P450c21 and the I172N protein expressed in Rat-1 cells were both distributed in the perinuclear space in a lacelike structure typical of ER location (Fig. 5). Therefore, ER localization did not seem to be affected by the mutation.
In addition to cytological examination, we also used a biochemical method to examine cellular localization of the I172N mutant protein. Cellular proteins were fractionated into the microsomal and soluble portions. As shown in Fig. 6A, wild type P450c21 sedimented in the pellet portion, indicative of its membrane association. Likewise, the I172N protein was also fractionated into the pellet portion. Therefore, the I172N mutant was associated with the membrane as well. There was no immunoreactivity from the yeast strain harboring vector pYE8 only.  Some peripheral membrane proteins can associate with the membrane not by direct membrane integration, but by charge interaction. These proteins can be extracted into the soluble fraction by salt in alkaline pH. To separate peripheral from integral membrane proteins, we translated proteins in vitro in the presence of microsomes, followed by alkaline extraction and centrifugation. Fig. 6B showed that proteins translated from both wild type plasmid WT-c21 and the mutant I172N still sedimented into the pellet after alkaline extraction, indicating tight membrane integration. Another ⌬52 protein of P450c21, which is truncated at the N terminus by 52 amino acids, could not integrate into the membrane and remained in the supernatant. This experiment further established the nature of the I172N mutant protein as an integral membrane protein. Therefore, the biochemical procedure, together with cytological detection, showed that the I172N mutant protein was localized in the ER through tight membrane integration. Since the Nterminal membrane targeting signal of I172N is intact, it is reasonable that this protein can still target into the ER membrane with equal efficiency.
Proteinase K Digestion Showed Altered Conformation of the I172N Mutant Protein-To probe the structure of mutant P450s, we digested P450c21 from yeast microsomes with increasing concentrations of proteinase K (Fig. 7). Wild type P450c21 was first digested into three major fragments at low proteinase K concentration (0.2 to 0.4 mg/ml). Then it was completely degraded at higher proteinase K concentration (1 mg/ml). This result is similar to that obtained from digesting purified bovine P450c21 from lipid vesicles by trypsin (35,36). It indicated that wild type P450c21 was folded into a structure with limited protease entry sites at its surface. Initial digestion at the surface of the protein generates a few proteolytic fragments, which were then further digested to completion after the disruption of the tertiary structure of the protein.
The I172N mutant protein isolated from yeast microsomes was present at a slightly lower amount (lane 6, Fig. 7). This protein was more sensitive to proteinase K digestion. It was digested to completion at a low concentration (0.2 mg/ml) when proteinase K just began to digest the wild type P450c21 into three fragments. The lack of visible bands in lane 8 of Fig. 7 was not due to the lower level of the I172N protein, as overexposure of the gel did not show any band. The complete sensitivity of the I172N protein toward proteinase K digestion indicated that it was partially unfolded so as to allow penetration of the protease into the protein. The I172N mutant expressed in COS-1 cells was also more sensitive to trypsin digestion than the wild type P450c21 (data not shown). These results indicate that the I172N mutant protein had partially unfolded conformation which could be detected by higher sensitivity toward proteinase K digestion, irrespective of the cell types which expressed the proteins.

DISCUSSION
The Ile-172 to Asn substitution is one of the most common mutations of the CYP21A2 gene causing congenital adrenal hyperplasia. This residue seems to be in a functionally important motif as its sequence is conserved in different microsomal P450s (Fig. 1). We have in this report characterized wild type and mutant P450c21 with point mutations in the vicinity of Ile-172, with respect to the amount of protein production, the rate of protein synthesis, enzymatic activity, and kinetic properties. Our data showed that replacing Ile-171 and -172 causes enzyme deficiency.
The I172N protein had drastically reduced enzymatic activity, which could be accounted for by its lower hemoprotein yield and altered catalytic parameters. Although the I172N protein was synthesized at the normal rate (Fig. 3) and its steady state level was similar to that of the wild type protein (Fig. 2), the amount of the heme-bound P450 form of the I172N protein was only 1/10 that of the wild type form (Table I). In addition, the I172N hemoprotein was obtained mostly as the P450 rather than the P420 form, if care was taken and substrate was added to stabilize the protein during purification. These results suggest that the decreased yield of the I172N hemoprotein was due to the decreased efficiency of the apoprotein to take up heme.
The I172N protein was partially unfolded as shown by its increased sensitivity toward proteinase K digestion. Many denatured proteins are known to be recognized by degradation enzymes (37,38) and tend to degrade faster than native proteins (39). The lower stability was also observed for a polymorphic form of P4502C13 with a single Ser-180 to Cys substitution (40). Therefore, the reduced amount of the I172N protein in the microsome appears to correlate with its sensitivity toward proteinase K digestion.
The I172N protein also had a much lower V max value (Table   FIG. 6. Membrane association of P450c21. A, fractionation. Yeast proteins from yeast cells harboring wild type P450c21 (15 g) or its I172N mutant (60 g) were fractionated into the particulate (P) and soluble portions (S) as described under "Experimental Procedures," electrophoresed, and reacted with anti-P450c21 antisera. Lane 1 contains lysate from cells harboring vector pYE8. B, membrane integration assay. Plasmids harboring wild type (WT-c21), I172N, or truncated (⌬52) forms of P450c21 cDNA were transcribed and translated in vitro in the presence of membranes. The total translation products (T) were extracted with alkali and centrifuged to separate into the supernatant (S) and pellet (P) forms. The full-length translation products are indicated by arrows. FIG. 7. Proteinase K digestion of wild type P450c21 and its I172N mutant expressed in yeast. Yeast microsomes (120 g of proteins) containing either wild type P450c21 or the I172N mutant protein was digested with various concentrations of proteinase K (from 0.02 to 1 mg/ml) as indicated on top of each lane at room temperature for 30 min. The digestion products were separated by gel electrophoresis before immunoblotting. I), which is another consequence of its conformational change. The combined effects of lower hemoprotein content and reduced catalytic function resulted in a defective protein with about 100-fold reduction in enzymatic activity.
There has been an earlier suggestion that the I172N mutant protein might be impaired in ER targeting (19). We showed that both normal P450c21 and its I172N mutant are localized to the ER membrane by a combination of methods including immunofluorescence (Fig. 5) and cellular fractionation (Fig. 6). This result is expected since the N-terminal membrane targeting and anchoring domain of the I172N mutant is intact. There is, however, speculation that the Ile-172 motif might be associated with the ER membrane, probably not by spanning the membrane but by forming a loop structure (8). Our data do not rule out the possibility that the Ile-172 motif might be involved in other types of membrane interaction. In addition to spanning the membrane by an ␣-helical domain, a protein can be associated with the lipid bilayer by direct fatty acylation (41,42), through a glycosylphosphatidylinositol anchor (43), or by noncovalent interaction with other membrane proteins. The exact nature of the interaction of the microsomal P450s with the ER membrane besides the N-terminal crossing is not known yet and should be investigated further.
A different mutant protein, I171N, also had lower enzymatic activity. This protein had a lower steady state level (Fig. 2), which was probably due to its lower synthetic rate (Fig. 3). Unlike I172N, the amount of the I171N P450 roughly correlated with the amount of apoprotein (Table I and Fig. 2). The V max value of the I171N P450 was only slightly lower (about 2/3) than that of the wild type protein, when the same amount of P450s were compared (Table I). It suggested that the defect of the I171N protein is the decreased amount and the slightly impaired catalytic rate.
We used mammalian and yeast cells as two separate expression systems to obtain wild type P450c21 and mutant proteins for our study. Most of the kinetic parameters presented in this report were obtained from proteins expressed in yeast, as yeast cells are easier to grow and higher amounts of P450c21 can be obtained more easily for the study. Some of the data were also confirmed using mammalian expression systems. Since similar results were obtained regarding the structure and function of the protein in spite of the expression system, different expression systems provide a cross-check for the experimental results. We are certain we were measuring the property of the protein, not any artifact resulting from the expression system.
To probe the structure of P450c21, we investigated the differential sensitivity of the wild type and mutant proteins toward proteinase K digestion. Protease digestion has been widely used to study the conformational change of a protein (44 -46). It, however, does not provide detailed structural information of the protein, which can be obtained only through crystallization and x-ray diffraction. What are the specific structural changes caused by a single substitution? How do these structural changes affect heme-binding capacity and catalytic activity? These are important questions which await further investigation.