Báo cáo khoa học: Cholesterol interaction with the related steroidogenic acute regulatory lipid-transfer (START) domains of StAR (STARD1) and MLN64 (STARD3) ppt

Cholesterol interaction with the related steroidogenic acute regulatory lipid-transfer (START) domains of StAR (STARD1) and MLN64 (STARD3) Julian Reitz 1 , Katja Gehrig-Burger 1 , Jerome F. Strauss III 2 and Gerald Gimpl 1 1 Institute of Biochemistry, Gutenberg-University Mainz, Germany 2 Department of Obstetrics & Gynecology, Virginia Commonwealth University, Richmond, VA, USA Cholesterol is an essential multifunctional lipid in most eukaryotic cells. It exerts a strong influence on the physical state of the plasma membrane, forms choles- terol–sphingolipid-rich microdomains such as caveolae and lipid rafts, is necessary for the activity of several membrane proteins, and serves as the precursor for steroid hormones [1–5]. Despite many efforts, the path- ways and mechanisms of cellular cholesterol trafficking are currently not well understood. Misfunctions of cholesterol transport are linked to a variety of diseases [6,7]. The biosynthesis of steroid hormones requires the transfer of cholesterol from multiple sources to the inner mitochondrial membrane, where steroidogenesis begins with the conversion of cholesterol to pregneno- lone. The translocation of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis, is mediated by steroidogenic acute regulatory protein (StAR, STARD1) [8–12]. The mechanism by which STARD1 moves cholesterol to the inner mitochondrial membrane is currently unclear [13]. Mutations that inactivate STARD1 in humans lead to an impaired ability of the adrenal gland to pro- duce steroid hormones, a potentially lethal disease known as congenital lipoid adrenal hyperplasia [14]. Ablation of the StarD1 gene in mice also causes impaired steroidogenesis and adrenal lipid accumula- tion [15]. STARD1 is synthesized as a 37 kDa phos- phoprotein with an N-terminal mitochondrial targeting sequence that is cleaved during mitochondrial entry (Fig. 1A). Deletion of 62 N-terminal residues (N-62 STARD1), including the leader peptide, resulted in a Keywords cholesterol; MLN64; STARD1; STARD3; START proteins Correspondence G. Gimpl, Institute of Biochemistry, Gutenberg-University Mainz, Becherweg 30, 55128 Mainz, Germany Fax: +49 6131 3925348 Tel: +49 6131 3923829 E-mail: gimpl@uni-mainz.de (Received 14 January 2008, revised 5 Febru- ary 2008, accepted 14 February 2008) doi:10.1111/j.1742-4658.2008.06337.x The steroidogenic acute regulatory (StAR)-related lipid transfer (START) domains are found in a wide range of proteins involved in intracellular trafficking of cholesterol and other lipids. Among the START proteins are the StAR protein itself (STARD1) and the closely related MLN64 protein (STARD3), which both function in cholesterol movement. We compared the cholesterol-binding properties of these two START domain proteins. Cholesterol stabilized STARD3-START against trypsin-catalyzed degrada- tion, whereas cholesterol had no protective effect on STARD1-START. [ 3 H]Azocholestanol predominantly labeled a 6.2 kDa fragment of STARD1-START comprising amino acids 83–140, which contains residues proposed to interact with cholesterol in a hydrophobic cavity. Photoaffinity labeling studies suggest that cholesterol preferentially interacts with one side wall of this cavity. In contrast, [ 3 H]azocholestanol was distributed more or less equally among the polypeptides of STARD3-START. Overall, our results provide evidence for differential cholesterol binding of the two most closely related START domain proteins STARD1 and STARD3. Abbreviations MLN64 (= STARD3), metastatic lymph node 64; MbCD, methyl-b-cyclodextrin; NBD-cholesterol, 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)amino]-23,24-bisnor-5-cholen-3-ol; SELDI, surface-enhanced laser desorption/ionization; StAR (= STARD1), steroidogenic acute regulatory protein; START, steroidogenic acute regulatory protein lipid-transfer domain. 1790 FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS cytosolic protein with full activity, as shown in intact cells and in isolated mitochondria [16–18]. The func- tionally active C-terminal domain of STARD1 con- tains the StAR-related lipid-transfer (START) domain. START domains consist of 200–210 amino acids and are found in a wide range of proteins involved in several cellular functions, including lipid transport, signal transduction, and transcriptional regulation [19]. Among the START proteins are the StAR protein itself (STARD1) and the closely related metastatic lymph node 64 (MLN64) protein (STARD3). Both proteins function as cholesterol-binding proteins [20,21]. Their START domains share 37% sequence identity. STARD3 is overexpressed in certain breast cancers [22]. The protein contains four transmembrane helices that target it to the membrane of late endosomes [23] (Fig. 1A). However, the physiological function of STARD3 is currently unclear. It may be involved in steroidogenesis in the human placenta, which lacks STARD1 [24,25]. The START domain at the C-termi- nal half of STARD3 is believed to be exposed to the cytosol. In its isolated form, STARD3-START is able to promote steroidogenesis even more efficiently than intact STARD3 [26]. The crystal structure of the unli- ganded START domain of human STARD3 has been resolved [20]. This structure shows a hydrophobic tun- nel that expands throughout the length of the START domain and is perfectly sized to accommodate a single cholesterol molecule [20]. A similar structure has been reported for the cholesterol-regulated START pro- tein 4 (STARD4) [27]. For another START protein, the phosphatidylcholine transfer protein (STARD2), it has been directly shown that the tunnel represents the binding site of the lipid, in this case phosphatidylcho- line [28]. To understand the molecular mechanism how cho- lesterol is transferred by STARD1 and STARD3, the cholesterol-binding sites of these proteins have to be identified. As a crystal structure of a cholesterol– START complex is not yet available, other methods are required to explore the cholesterol–protein interac- tion. One approach is molecular modeling based on the knowledge of the unliganded STARD3 structure. Two such modeling studies have been recently per- formed for the START domains of STARD1 and STARD3 [29,30]. This led to the proposal that STARD1-START shuttles cholesterol carried in its hydrophobic cavity between the outer and inner mito- chondrial membranes [20]. However, spectral and bio- chemical data supported the view that STARD1 partially unfolds and forms molten globules in the low-pH environment of the outer mitochondrial membrane. These intermediates were hypothesized to facilitate the cholesterol transfer of STARD1 to the mitochondrial inner membrane through a mechanism that does not involve sterol shuttling [31,32]. A START N C START N C B 123 97 66 45 31 21 14 – – – – – – C m/z 20000 25000 30000 35000 m/z 20000 25000 30000 35000 Intensity Intensity 0 5 10 15 20 0 10 20 30 40 50 29162.8+H 26167.8+H STARD1-START STARD3-START Fig. 1. Expression of the START domains of STARD1 and STARD3. (A) Domain organization of the START proteins STARD1 (285 amino acids) and STARD3 (445 amino acids). Both proteins possess a ste- rol-binding START domain ( 200 amino acids) in their C-terminal regions. The N-terminal targeting sequence of STARD1 is cleaved upon entry into the mitochondria, and is nonessential for the activity of STARD1 [16–18]. The N-terminal part of STARD3 possesses four transmembrane segments that target the protein to late endosomes. The START domain in STARD3 is exposed to the cytosol and is func- tionally active in its isolated form [26]. (B) Purification of the START domains of STARD1 and STARD3 expressed in Escherichia coli. The proteins were purified from E. coli, resolved by SDS ⁄ PAGE, and identified by Coomassie blue staining. Lane 1: marker. Lane 2: STARD1-START (2 lg of protein). Lane 3: STARD3-START (6 lgof protein). (C) SELDI-TOF of STARD1-START and STARD3-START. J. Reitz et al. Cholesterol binding of START proteins FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS 1791 Here, we analyzed the cholesterol-binding character- istics of the two most related START proteins, STARD1 and STARD3. Photoaffinity labeling with radiolabeled 6-azocholestanol as the photoreactive cho- lesterol probe was employed to characterize and com- pare the cholesterol binding of the START domains. This cholesterol analog (previously often termed photocholesterol) has already been successfully applied for various proteins [23,33–36]. Overall, this study addresses the question of whether or not the related START domains of StARD1 and StARD3 interact with cholesterol in a similar manner. Results Expression of the START domains The recombinant START proteins each contain a His 6 - tag at their C-terminus. The proteins were expressed in BL21 Escherichia coli and purified by affinity chroma- tography using an Ni 2+ –nitrilotriacetic acid agarose matrix. Figure 1B shows the Coomassie stains of the purified proteins. The apparent molecular masses of the His-tag START proteins in the SDS ⁄ PAGE system were slightly greater than the calculated molecular masses of 25 769 Da (pI 6.42) and 26 847 Da (pI 8.43) for STARD1-START and STARD3-START, respectively (Fig. 1B). This discrepancy has also been observed by Arakane et al. [17] in the case of STARD1-START. To explore this issue, we also determined the molecular masses of both START proteins by surface-enhanced laser desorption/ionization (SELDI)-TOF MS. Molecu- lar masses of 26 167 and 29 162 Da were found for STARD1-START and STARD3-START, respectively (Fig. 1C). Whereas the molecular mass of STARD1- START is relatively close (+398 Da) to the calculated value of 25.7 kDa, the mass of STARD3-START is about 2.3 kDa higher than that calculated for the unmodified polypeptide. This could reflect post-transla- tional protein modification. The expression levels of STARD1-START and STARD3-START were similar. Cholesterol binding of the START proteins In order to verify the cholesterol binding of the START proteins, we used the fluorescent cholesterol reporter 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]- 23,24-bisnor-5-cholen-3-ol (NBD-cholesterol). This cho- lesterol analog has successfully been employed to analyze the cholesterol binding of STARD1-START [21,31]. A strong increase in the fluorescence intensity of NBD-cholesterol occurs when the ligand binds to the hydrophobic environment of the START proteins. This has recently been studied in detail by Petrescu et al. [21] in the case of STARD1-START. The binding of NBD-cholesterol to each of the START proteins shows a saturating profile (supplementary Fig. S1A,B). The curves were fitted using a nonlinear regression algorithm according to one-site models, and yielded K D values of 161 ± 45 nm (n = 3) for STARD1-START and 58 ± 16 nm (n = 3) for STARD3-START. Thus, STARD3-START bound NBD-cholesterol with a slightly higher affinity than did STARD1-START. Two-site models did not result in significantly better fittings of the binding data. According to one model of START domain action, a pH-dependent molten globule transition of STARD1 is required for sterol transfer activity at the level of the mitochondrial outer membrane [31,32]. Therefore, we also measured the fluorescence of NBD-cholesterol (500 nm) bound to STARD1-START (10 nm)atan acidic pH. At pH 3, the sterol binding of STARD1- START was about three-fold lower than the sterol binding measured at pH 7.4 (data not shown). Analysis of the stabilizing effect of cholesterol on START proteins Cholesterol and its analogs are able to stabilize pro- teins against proteolysis or thermal degradation [37]. To test whether this occurs in the case of the START proteins, we analyzed the migration behavior of these proteins in SDS gels under various conditions. First, the START proteins were incubated (for 20 min at 25 °C) in the presence of cholesterol, photo- cholesterol, or buffer control. The proteins were irradi- ated with UV light for 10 min prior to separation by SDS ⁄ PAGE, western blotting, and immunodetection with antibody to His (supplementary Fig. S2A). It is important to note that the His-tag is localized at the C-terminus of both proteins, so that only molecular species with an intact C-terminus are visible on the immunoblots. The immunoblot revealed no significant differences among treated and untreated START pro- teins. Faint staining was observed for the putative dimer forms of the proteins in addition to the predom- inant monomer ( 30 kDa) bands. We did not find a slight increase in the molecular size of the START pro- teins in the photoactivated samples of the photocholes- terol-containing samples. Most probably, the labeled species is below the detection limit, due to the low photoaffinity yield (< 9%). We next analyzed the resistance of the START pro- teins to degradation in the presence and absence of cholesterol. The proteins were pretreated either with buffer solution or cholesterol–methyl-b-cyclodextrin Cholesterol binding of START proteins J. Reitz et al. 1792 FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS (MbCD) (0.1 mm) for 20 min at 25 °C. Then, the sam- ples were incubated for increasing times (6 h, 24 h, 80 h) at 40 °C prior to separation by SDS⁄ PAGE, western blotting, and immunodetection with antibody to His (supplementary Fig. S2B). For STARD1- START, we did not observe any evidence of degrada- tion during the time course of this experiment. In contrast, in the case of STARD3-START, an addi- tional band with a slightly decreased apparent molecu- lar mass (by  3–4 kDa) appeared after an incubation period of 24 h or longer. The presence of cholesterol did not influence the appearance of this additional band (supplementary Fig. S2B). When the samples were treated with trypsin (10 min or 40 min at 37 °C), additional bands were observed on the immunoblots for both START proteins (Fig. 2). Two additional molecular species with slightly higher electrophoretic mobilities appeared for STARD1-START. The presence of cholesterol did not inhibit the appearance of these additional bands, nor did it affect the protein patterns of the immunoblots. STARD3-START was more sensitive to trypsinolysis (Fig. 2). When trypsin was incubated for 40 min, most of the STARD3-START was either totally degraded or, more probably, had its C-terminus bearing the His- tag cleaved. Incubations with trypsin for more than 60 min resulted in immunoblots with no detectable START proteins (not shown). However, cholesterol was clearly able to inhibit the trypsinolysis of STARD3-START (Fig. 2). Cholesterol labeling of STARD1-START To determine the cholesterol docking site within the START domains of STARD1 and STARD3, we per- formed photoaffinity labeling with [ 3 H]photocholester- ol and subsequent chemical or enzymatic cleavage of the photoactivated samples. Highly reproducible frag- mentation patterns were obtained when the protein was subjected to chemical cleavage by cyanogen bromide (CNBr), which hydrolyzes peptide bonds C-terminal to Met residues. The predicted cleavage products are listed in Table 1 for STARD1-START. In the case of STARD1-START, the [ 3 H]photocholes- terol radiolabel was incorporated nearly quantitatively into a single band at about 6.2 kDa (Fig. 3). Even when we increased the protein amounts from 20 lg (Fig. 3, filled symbols) to 60 lg (Fig. 3, open symbols), the label was predominantly incorporated in a  6.2 kDa fragment. A control labeling of STARD1- START with [ 3 H]photocholesterol but without UV irradiation did not reveal any bands (Fig. 3, dia- monds). Similarly, when cholesterol was added to the samples at a ‡ 50-fold molar excess over [ 3 H]photo- cholesterol, the appearance of the  6.2 kDa fragment ++++–++++–Try +–+––+–+––Cho 40´10´40´10´ STARD3-STARTSTARD1-START 31– Fig. 2. Stability of the START domains of human STARD1 and STARD3 in the presence or absence of cholesterol. The START pro- teins (1 lgÆlL )1 ) were preincubated with buffer solution or choles- terol-MbCD (Cho) (0.1 m M) for 20 min at 25 °C. Then, the samples were incubated in the presence of trypsin (Try) for 10 min or 40 min at 37 °C. The proteins were precipitated with acetone, dis- solved in water, separated by SDS ⁄ PAGE, and subjected to wes- tern blotting, using antibody to His and Amersham ECL Plus for detection. Table 1. Cleavage and fragmentation of STARD1-START by CNBr. The molecular mass data are calculated average masses [M +H] + according to the program PEPTIDE MASS (Expasy). Molecular mass (Da) Residues Sequence 102.1 1 M 2300.4 2–21 EETLYSDQELAYLQQGEEAM 2885.2 22–47 QKALGILSNQEGWKKESQQDNGDKVM 2294.7 48–68 SKVVPDVGKVFRLEVVVDQPM 1419.6 69–79 ERLYEELVERM 302.3 80–82 EAM 6236.2 83–140 GEWNPNVKEIKVLQKIGKDTFITHELAAEAAGNLVGPRDFVSVRCAKRRGSTCVLAGM 707.7 141–147 DTDFGNM 1705.9 148–163 PEQKGVIRAEHGPTCM 7554.7 164–229 VLHPLAGSPSKTKLTWLLSIDLKGWLPKSIINQVLSQTQVDFANHLRKRLESHPASEARCHHHHHH J. Reitz et al. Cholesterol binding of START proteins FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS 1793 was suppressed (not shown). A predicted fragment of this size (6236 Da) corresponds to STARD1-START residues 83–140, as listed in Table 1. Owing to partial cleavage, CNBr fragments with sizes similar to the 6236 Da species are possible, such as the combined fragments with molecular masses of 5185 Da (= 2300 + 2885 Da), 5179 Da (= 2885 + 2294 Da), and 6598 Da (= 2885 + 2294 + 1419 Da). To deter- mine whether partially cleaved fragments are present within this molecular range, we performed MS (see inset in Fig. 3). The sample for SELDI-TOF MS was prepared as described for STARD1-START, except that unlabeled photocholesterol was used instead of [ 3 H]photocholesterol. In the mass spectrum, two major peaks are observed within the molecular range 4000– 7000 m ⁄ z, a 5194 Da species and a 6263 Da species. The 5194 Da species could represent either the com- bined 5185 Da fragment or the (possibly oxidized) par- tially uncleaved 5179 Da fragment. The 6263 Da peak should represent the 6236 Da fragment, perhaps modi- fied by formylation (+26 Da). Covalent coupling of one molecule of photocholesterol should add a mass of about 386 Da to the 6236 Da fragment, resulting in a  6.6 kDa species. A small shoulder area to the right to the 6263 Da peak (Fig. 3, inset) might include such a species. However, a partial uncleaved 6598 Da frag- ment (see above) would overlap with this species and does not allow us to reach a definite conclusion on this point. STARD1-START protein labeled with photo- cholesterol and cleaved by CNBr did not reveal sub- stantial differences in the mass spectra in comparison with samples untreated with photocholesterol prior to cleavage with CNBr, probably because of the low photoaffinity yield (< 9%), which results in the labeled species being below the detection limit. Affinity labeling with [ 3 H]photocholesterol and subsequent CNBR cleavage were carried out for STARD1-START at neutral and acidic pH. Typical fragmentation profiles are demonstrated in Fig. 4A (at neutral pH) and Fig. 4B (at acidic pH). Quantitation of the results is shown in Table 2. Cholesterol labeling of the 6.2 kDa fragment was lower at pH 3.0 than at pH 7.4. Moreover, in gel slices at and close to the gel front, a markedly higher incorporation of radioactivity was found at acidic pH than at neutral pH. These gel slices contain oligopeptide fragments with molecular masses < 2 kDa, including unbound [ 3 H]photocholes- terol. According to the fragmentation pattern (Table 1), these could represent peptides with molecular masses of 1705, 751, and 302 Da. Obviously, at pH 3, the choles- terol labeling of STARD1-START is less specific than the labeling at pH 7.4. Cholesterol labeling of STARD3-START In case of STARD3-START, photoaffinity labeling with [ 3 H]photocholesterol and subsequent CNBr cleav- age revealed several peaks, which were numbered from 1 to 5 (Fig. 5, circles). The predicted cleavage products for STARD3-START are listed in Table 3. Peak 1 cor- responds to molecular mass > 26.6 kDa, and should represent uncleaved STARD3-START. Peaks 2 and 3 can be assigned to the predicted fragments of 13 262 Da (residues 93–212) and 10 556 Da (resi- dues 1–92), respectively (Table 3). Peak 4 corresponds to the fragment of size 2972 Da (residues 213–236). Peak 5 represents unbound [ 3 H]photocholesterol (Fig. 5, dotted line). SELDI-TOF of CNBr-cleaved STARD3-START revealed major peaks oat 3187, 11 575, 14 332, and 25 918 Da, and a minor peak at 29 152 Da (not shown). The 25 918 Da species ( 11 575 + 14 332 Da) should be partially cleaved polypeptide. Thus, each of the masses of the three Gel slice number 0 102030405060708090100 Radioactivity (dpm) 0 5000 10 000 15 000 20 000 25 000 30 000 35 000 26.6 17.0 14.4 6.5 3.5 1.4 m/z 4000 5000 70006000 Intensity 0 2 4 6 8 6263.1+H 5194.9+H Fig. 3. Cholesterol labeling and chemical cleavage of STARD1- START. STARD1-START (20 lg of protein, filled circles and dia- monds, and 60 lg of protein, open circles) was incubated with [ 3 H]photocholesterol (50 lM) for 20 min at 25 °C. Then, the sam- ples were either UV-irradiated (circles) or not UV-irradiated (control, diamonds) for 10 min at 4 °C. The protein was precipitated with acetone, dissolved in water, and subjected to chemical cleavage by CNBr for 24 h at 37 °C. The proteins were separated by SDS ⁄ PAGE. The gel was cut into 1 mm slices and incubated over- night at room temperature with a scintillation cocktail. The radioac- tivity of each slice was counted. The molecular mass (in kDa) was estimated from a control lane loaded with molecular size markers, and is given at the top of each panel. The reference line (dotted) corresponds to unbound [ 3 H]photocholesterol. The inset shows a SELDI-TOF mass spectrum of STARD1-START cleaved by CNBr in (and calibrated for) the mass range 4000–7000 m ⁄ z. The sample for MS was prepared as described, except that unlabeled photo- cholesterol was used instead of [ 3 H]photocholesterol. Cholesterol binding of START proteins J. Reitz et al. 1794 FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS fragments is higher (215–1070 Da) than calculated for the corresponding unmodified polypeptide. This sug- gests that unknown post-translational protein modifi- cations are more or less equally distributed along the length of the protein. In control experiments in the presence of an excess of unlabeled cholesterol, low amounts of radioactivity were detected in the gel slices over the whole length of the gel (except at peak 5, cor- responding to unbound photocholesterol) (Fig. 5, dia- monds). Similar low amounts of radioactivity were observed when the START protein was denaturated by heat (5 min at 95 °C) (not shown). Discussion We have explored the cholesterol binding of the START domains of the two most related START pro- teins, STARD1 and STARD3. Both proteins bound the fluorescent cholesterol reporter NBD-cholesterol with high affinity. With respect to the sterol binding of STARD1-START, our results were within the range previously reported [21]. Cholesterol is able to stabilize proteins, e.g. by protecting them from thermal dena- turation or proteolytic degradation, as shown for the oxytocin receptor [37], the Torpedo californica acetyl- choline receptor [38], and rhodopsin [39]. When STARD3-START was incubated for many hours (24– 80 h) at 40 °C, an additional band (truncated by  3 kD in apparent molecular mass) appeared in immunoblots. This additional molecular species could represent either a denaturated form of the protein with higher electrophoretic mobility or an N-terminal trun- cated fragment of STARD3-START resulting from cleavage by a protease still present in our preparation. In each case, the presence of cholesterol was not able to suppress the appearance of this additional molecular species. However, cholesterol had a protective effect against the trypsinolysis of STARD3-START, whereas the cleavage of STARD1-START was not affected. Both START proteins possess several cleavage sites Table 2. Efficiency of labeling of the 6.2 kDa fragment with [ 3 H]photocholesterol in STARD1-START. Labeling was performed with [ 3 H]photocholesterol (50 lM) and STARD1-START (5 lM). The samples were UV-irradiated for 10 min at 4 °C at the indicated pH in a volume of 100 lL. The protein was precipitated with acetone, dissolved in water, and subjected to chemical cleavage by CNBr for 24 h at 37 °C. The proteins were separated by SDS ⁄ PAGE. The gel was cut into 1 mm slices. The slices were incubated with scintillation cocktail, and the radioactivity of each slice was counted. To calculate the labeling efficiency, the radioactivity in the peak area ( 15 slices) corresponding to a molecular mass of 6.2 kDa was integrated. Control samples were treated under the same conditions except for the UV crosslinking step. These control values (integrated radioactivity of  15 slices corresponding to a molecular mass of 6.2 kDa) were subtracted from the sample data. Labeling efficiency is the amount of [ 3 H]photocholesterol incorporated into the 6.2 kDa fragment of STARD1-START (0.5 nmol), with 100% being equal to 0.5 nmol of the photolabel. The data are means ± SD (n = 3). To obtain the relative labeling efficiencies, the data were normalized to 100%. Membranes Labeling efficiency (%) Relative efficiency (%) STARD1-START, pH 7.4 8.8 ± 1.9 100.0 ± 21.5 STARD1-START, pH 3.0 5.6 ± 2.2 63.6 ± 25.0 Gel slice number Radioactivity (dpm) Radioactivity (dpm) 0 15 000 A Gel slice number 0 102030405060708090100 0 102030405060708090100 0 15 000 B pH 7.4 pH 3.0 * * Fig. 4. Cholesterol labeling and CNBr cleavage of STARD1-START at different pH values. The START proteins (each 20 lg of protein) were incubated with [ 3 H]photocholesterol (50 lM) for 20 min at 25 °C at pH 7.4 (A) or pH 3.0 (B). Then, the samples were UV-irradiated for 10 min at 4 °C. The protein was cleaved by CNBr and further processed as described in the legend for Fig. 3. The asterisks mark the position of the 6.2 kDa band. The reference lines (dotted) correspond to the gel front line containing unbound [ 3 H]photocholesterol and fragments of less than  1 kDa. J. Reitz et al. Cholesterol binding of START proteins FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS 1795 (Arg and Lys residues) for trypsin within their N-ter- minal sequence, which could lead to the observed frag- mentation pattern. One simple explanation of the data is that the N-terminal region of STARD3-START directly interacts with cholesterol, thus impeding the access of trypsin. Alternatively, cholesterol could stabi- lize a conformation of the protein that is more resis- tant to trypsinolysis. What is known about the cholesterol-binding site of the START domains of STARD1 and STARD3? The crystal structure of human STARD3-START revealed an a ⁄ b-fold consisting of a nine-stranded twisted b-sheet and four a-helices [20]. The START domains of STARD3 [20], STARD4 [27], phosphatidylcholine transfer protein [28,40], and related bacterial proteins share this basic structure [41,42]. A STARD1-START model based on the structure of STARD3-START is shown in Fig. 6A,B in two views. The view in Fig. 6B is related to that in Fig. 6A by a  90° rotation about the y-axis. The b-strands in the order b 1 –b 2 –b 3 –b 9 –b 8 – b 7 –b 6 –b 5 –b 4 form a U-shaped unclosed b-barrel with a predominant hydrophobic cavity that is optimally sized to bind a single cholesterol molecule (Fig. 6A). The roof of the cavity is mainly formed by the C-terminal a 4 -helix. The access of cholesterol to this cavity may be enabled by conformational changes of the a 4 -helix and the adjacent loops. In the case of STARD1- START, we have identified a 6.2 kDa fragment comprising amino acids 83–140 as a major cholesterol- binding site (Fig. 7, residues 83–140, highlighted in gray). The corresponding structures, colored yellow in Fig. 6A,B, are the b-strands b 7 –b 6 –b 5 –b 4 including the W 3 -loop (connecting b 5 and b 6 ) and part of the a 3 -helix. This suggests that cholesterol bound in the cavity is preferentially in contact with one side wall of this cavity. The geometry of the cavity in STARD1- START is well suited for a ligand with the size and shape of cholesterol [29,30]. Critical residues proposed to interact with cholesterol are localized within the fragment containing amino acids 83–140. These resi- dues are in magenta in Fig. 6B. For example, the acidic side chain of Glu107 in STARD1-START (Glu169 in STARD1) (corresponding to Asp117 in STARD3-START) was proposed to be involved in specific cholesterol binding, most likely with the 3b-hydroxyl group of cholesterol [20]. Cholesterol might also interact with the conserved and buried Arg residue at position 126 in STARD1-START (Arg136 in STARD3-START) [20]. The charged residues Glu107 and Arg126 in human STARD1-START, which are equivalent to Glu168 and Arg187 in the hamster STARD1 model, were found to form a salt bridge at the bottom of the hydrophobic pocket of the START domain [29,30]. In STARD3-START, these residues may interact with the 3b-hydroxyl group of cholesterol via hydrogen bonding to an included water molecule [30], as was concluded from molecular Gel slice number 0 10203040506070 Radioactivity (dpm) 0 1000 2000 3000 4000 5000 6000 7000 26.6 17.0 14.4 6.5 3.5 1.4 1 2 3 4 5 Fig. 5. Cholesterol labeling and chemical cleavage of STARD3- START. The protein (20 lg) was incubated with [ 3 H]photocholes- terol (50 l M) for 20 min at 25 °C. As a control, STARD3-START (20 lg) was incubated with [ 3 H]photocholesterol (50 lM) in the presence of a 50-fold molar excess of cholesterol (diamonds). Then, the samples were UV-irradiated, cleaved by CNBr, and further processed as described in the legend for Fig. 3. The molecular mass (in kDa) was estimated from a control lane loaded with molecular size markers, and is given at the top of panel. The reference line (dotted) corresponds to unbound [ 3 H]photocholesterol. Table 3. Cleavage and fragmentation of STARD3-START by CNBr. The molecular mass data are calculated average masses [M +H] + according to the program PEPTIDE MASS (Expasy). Molecular mass (Da) Residues Sequence 10 555.7 1–92 GSDNESDEEVAGKKSFSAQEREYIRQGKEATAVVDQILAQEENWKFEKNNEYGD TVYTIEVPFHGKTFILKTFLPCPAELVYQEVILQPERM 13 262.2 93–212 VLWNKTVTACQILQRVEDNTLISYDVSAGAAGGVVSPRDFVNVRRIERRRDRY LSSGIATSHSAKPPTHKYVRGENGPGGFIVLKSASNPRVCTFVWILNTDLKGRLPRYLIHQSLAATM 2972.3 213–236 FEFAFHLRQRISELGARAHHHHHH Cholesterol binding of START proteins J. Reitz et al. 1796 FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS modeling and structure-based thermodynamics [29,30]. Water molecules were in fact discovered inside the STARD3 crystal [20]. The replacement of the two charged residues Glu107 and Arg126 in STARD1- START by hydrophobic residues of similar volume resulted in the total loss of STARD1 activity [30]. According to molecular modeling, another residue located within the 6.2 kDa fragment could be involved in cholesterol interaction: Leu137 (Leu199) in STARD1-START (STARD1), and the corresponding Ser147 (Ser362) in STARD3-START (STARD3) [29,30]. In STARD1-START, cholesterol might contact Leu137 indirectly, mediated by at least one water mol- ecule, whereas in STARD3-START cholesterol was suggested to form a direct hydrogen bond with Ser147 [29,30]. Nevertheless, the major contributions to the C N β 4 α 1 α 4Ω 3 Ω 2 Ω 1 β 5 β 6 β 7 β 1 β 2 β 3 α 2 α 3 β 8 β 9 N C E L R A B Fig. 6. Model of STARD1-START. The model was build after sequence alignment of STARD1-START with STARD3-START, for which a crys- tal structure is known [20]. For a better depiction of the elongated hydrophobic pocket, the same ribbon diagram is displayed from two different views [(A) and (B)] using the program CHIMERA [51]. The view in (B) is related to that in (A) by a 90° rotation about the y-axis. The photocholesterol docking region is shown in yellow, and comprises half of the a 3 -helix and the strands b 3 –b 7 , including their connecting loops. The residues Glu107 (E), Arg126 (R) and Leu137 (L) (all marked in magenta) are located within this region and have been proposed to interact with cholesterol (see Discussion). Otherwise, the model is colored according to the secondary structure, with helices in red, b-strands in green, and loops in gray. Fig. 7. Alignment of the START domains of human STARD1 and STARD3. Sequence identities are marked by a star, and residues contribut- ing to the tunnel in STARD3 are marked in bold. STARD1 missense mutations causing congenital adrenal hyperplasia are underlined. The numbering of residues within the whole sequences of STARD3 and STARD1, respectively, is in parentheses. STARD1-START and STARD3- START share 37% sequence identity and  60% amino acid similarity. Residues 83–140, corresponding to the photocholesterol-interacting fragment in STARD1-START, are marked in bold and highlighted in gray. J. Reitz et al. Cholesterol binding of START proteins FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS 1797 energy of cholesterol binding are most likely provided by nonpolar contacts with side chains lining the hydro- phobic cavity of STARD1-START [29]. In contrast to STARD1-START, STARD3-START did not show preferential incorporation of photocho- lesterol into a single polypeptide. If one assumes the same cholesterol-binding site as in STARD1-START, one should expect that photocholesterol is primarily incorporated into the CNBr fragment 93–212. How- ever, this was clearly not the case. Instead, cholesterol labeling of STARD3-START was distributed more or less equally among the three fragments. This could indicate that the cholesterol molecule localized within the binding pocket of STARD1-START possesses a lower degree of freedom than the cholesterol molecule inside the tunnel of STARD3-START. Although both START domains show high structural similarity, a recent modeling approach provided evidence for slight differences in the orientation of the cholesterol ring within their cavities that may result in distinct contact sites for photocholesterol [29]. How is the nearly solvent-inaccessible cavity opened or closed in response to cholesterol loading and release? Access into the cavity is mainly occluded by the C-terminal a 4 -helix and the adjacent loops (Fig. 6A). Conformational changes of the amphipathic a 4 -helix allow opening of the cavity. This scenario is supported by spectroscopic measurements demonstrat- ing a loss of helical structure in STARD1 after binding of the cholesterol reporter NBD-cholesterol [21]. The a 4 -helix is believed to contact the phospholipid bilayer of the outer mitochondrial membrane [43]. According to one hypothesis, STARD1 thereby undergoes an acid-inducible structural change to a molten globule state [44]. Biophysical data provided evidence for a stronger association of STARD1 with the mitochon- drial outer membrane (e.g. with the protonated phos- pholipid head groups) at an acidic pH ( 3.5) [45]. We show here that under acidic pH conditions, the efficiency in photocholesterol labeling of STARD1- START was significantly but not dramatically decreased. Thus, a putative molten globule state of STARD1-START might be slightly more capable of releasing its bound cholesterol. However, the STARD1-mediated translocation of cholesterol into the mitochondria is not well understood. Probably, STARD1 acts in concert with other proteins, such as STARD4 and the peripheral benzodiazepine receptor, to transfer cholesterol from the outer to the inner membrane of the mitochondrion [43,46]. Taken together, our observations provide evidence for differential cholesterol interactions with the two most closely related START proteins. The importance of the cholesterol-binding site in STARD1-START is underlined by the fact that several disease-related mutations or truncations in human STARD1 appear to correspond to residues lining the interior of the hydrophobic cavity, or in the C-terminal a-helix, when mapped onto the STARD3-START structure [14,18,47]. However, it is important to mention that any con- clusions drawn from studies employing cholesterol analogs such as NBD-cholesterol or photocholesterol have to be judged with caution [35]. For example, photocholesterol is structurally different from choles- terol, having, associated with the B-ring, an additional ring structure consisting of two nitrogen atoms, and could be involved in significantly different interactions (e.g. hydrogen bonding) with certain amino acid side chains. Thus, it cannot be excluded that the difference in photocholesterol binding does not truly reflect a dif- ference in binding of native cholesterol. An ultimate understanding of the interaction of cholesterol with START proteins requires the structure(s) of choles- terol-occupied START proteins. Experimental procedures Expression of the START domains The recombinant START proteins were produced in BL21 E. coli expressing human STARD3-START (amino acids 216–445) [26], or N-62-STARD1 (STARD1-START), as previously described [17]. Each of the expressed proteins contained a His 6 -tag at the C-terminus. The bacteria were cultivated in LB medium containing 25 lgÆmL )1 kanamycin for STARD1-START or 25 lgÆmL )1 ampicillin for STARD3-START. For expression of the proteins, 400 mL of medium (with antibiotic) was inoculated with 1 mL of overnight culture. The medium was shaken at 37 °C until an attenuance of 0.5–1.0 at 600 nm was achieved. Expression was induced by the addition of 0.5 m isopropyl-b-d-thio- galactopyranoside. After 4.5 h, the bacteria were pelleted. The pellet was resuspended on ice in 10 mL of the fol- lowing buffer: 300 mm NaCl, 50 mm NaH 2 PO 4 ,20mm Tris ⁄ HCl (pH 7.4), and 10 mm b-mercaptoethanol. The bacteria were sonicated on ice (3 · 15 pulses of 1 s, output level 7), using a Branson Sonifier 250 (Branson, Danbury, CT, USA). The suspension was centrifuged at 4 °C for 30 min at 20 000 g (J2-21-centrifuge; Beckman, Munich, Germany). The supernatant was incubated with 500 lLof Ni 2+ –nitrilotriacetic acid–agarose matrix (Qiagen, Hilden, Germany). The mixture was rotated at 4 °C overnight. The matrix was placed in a column and washed with 20 mL of the following buffer: 300 mm NaCl, 50 mm NaH 2 PO 4 (pH 8.0), and 20 mm imidazole. STARD1-START was eluted with 2 mL of the following buffer: 300 mm NaCl, Cholesterol binding of START proteins J. Reitz et al. 1798 FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS 50 mm NaH 2 PO 4 (pH 8.0), and 250 mm imidazole. To avoid aggregation of STARD3-START, the STARD3 elu- tion buffer contained 40% (w ⁄ v) glycerol. The eluted pro- teins were dialyzed (molecular mass cutoff 12 kDa; Sigma, Schnelldorf, Germany) against the following buffer: 50 mm KCl, 50 mm Hepes (pH 7.4), and 1 mm dithiothreitol. For dialysis of STARD3-START, the following buffer was used: 150 mm NaCl, 50 mm KCl, 50 mm Tris (pH 7.4), 10 mm dithiothreitol, and 40% (w ⁄ v) glycerol. Immunoblotting Proteins were separated by SDS ⁄ PAGE and were trans- fered onto a nitrocellulose membrane using a tank blot sys- tem. Immunodetection was performed with appropriate antibodies: mouse anti-His serum (1 : 2000) and mouse anti-peroxidase Ig (1 : 1000). The proteins were detected with Amersham ECL Plus (GE Healthcare Life Sciences, Munich, Germany). The results were displayed and docu- mented using a VersaDoc 3000 imaging system (Bio-Rad, Munich, Germany). Photoaffinity labeling Photoaffinity labeling of the START proteins was performed using the photoreactive cholesterol analog [ 3 H]6,6-azocho- lestanol (termed [ 3 H]photocholesterol). [ 3 H]Photocholesterol was synthesized according to an established protocol [48]. Twenty micrograms of protein in a final volume of 200 lL were incubated with [ 3 H]photocholesterol (50 lm, 30–185 GBqÆmmol )1 ) for 20 min at room temperature. The sterol was complexed with MbCD (0.6 mgÆmL )1 ). For UV irradiation, either a 200 W Hg-lamp (k 330 nm; Leitz, Wetzlar, Germany) or a Transilluminator 4000 (Stratagene, Heidelberg, Germany) was used. The distance between the lamp of the Transilluminator and the samples was about 5 cm. During the irradiation, the samples were incubated on ice in 1.5 mL reaction tubes. The samples were irradiated for 10 min. When the 200 W Hg-lamp was used, the samples were irradiated in a cooled quartz cuvette with a magnetic stir-bar. The crosslinking efficiency obtained with the Trans- illuminator was found to be similar to that obtained with the 200 W Hg-lamp. The proteins were precipitated with 1 mL of cold acetone ()20 °C). The sample was stored at )20 °C for at least 1 h. The proteins were pelleted by centrifugation at 20 000 g for 10 min at 4 °C. The supernatant was removed. The pellet was dried with gaseous N 2 . The protein pellets were subjected to SDS ⁄ PAGE or to chemical or enzymatic cleavage. Cleavage of proteins For chemical cleavage, CNBr (Fluka, Germany) was used. The pellet (20 lg of protein) was resuspended in 30 lLof H 2 O. Seventy microliters of formic acid containing 100 lg of CNBr were added. The sample was incubated for 24 h at 37 °C in the dark. The solvent was evaporated with gaseous N 2 . For enzymatic cleavage, the protease LysC (Roche, Germany) was used. The pellet (20 lg of protein) was resuspended in 20 lL of the following buffer: 100 mm NH 4 HCO 3 (pH 8.5). One microgram of LysC in 1 lLof the same buffer was added, and the sample was incubated at 37 °C for 24 h in the dark in a gaseous N 2 atmosphere. SDS ⁄ PAGE To determine the molecular masses of the proteins, the Laemmli protocol was employed. For the separation of small protein fragments, the method described by Schaegger and von Jagow [49] was used. Scintillation counting The fragments of the labeled and cleaved proteins were sep- arated by tube gels (100 mm in length, 4 mm in diameter) or slab gels (50 mm in length, 1.5 mm in thickness). The gels were cut into 1 mm slices. Each slice was incubated overnight at room temperature in a scintillation vial (Canb- erra Packard, Dreieich, Germany) with 4 mL of the follow- ing scintillation cocktail: 90% (v ⁄ v) Lipoluma; 9% (v ⁄ v) Lumasolve; and 1% (v ⁄ v) H 2 O (Lumac-LSC; Perkin-Elmer, Groningen, the Netherlands). For scintillation counting, a Tri-Carb 2100 TR-counter (Packard, Dreieich) was used. Fluorescence spectroscopy The fluorescent cholesterol reporter NBD-cholesterol was used to verify the cholesterol binding of STARD1-START and STARD3-START. The measurements were performed with a Photon Technologies International (Birmingham, NJ, USA) spectrofluorometer (Quantamaster). The proteins were diluted with 25 mm potassium phosphate buffer (pH 7.4) including 0.0002% Tween-20 to a final concentration of 10 nm. The sample was transferred in a quartz cuvette that was placed in a cuvette holder equipped with a magnetic stir- bar. The sterol was added from ethanolic stock solutions. The samples were incubated for 10 min at 37 ° C before the fluorescence was recorded at constant temperature (37 °C). NBD-cholesterol was excited at 473 nm. Fluorescence emis- sion was monitored at 530 nm. Excitation and emission bandpasses were set to 4 nm. To reduce light scatter, a cutoff filter (495 nm) was placed in the emission path. The binding data were calculated using sigmaplot (version 8.0). MS A SELDI-TOF mass spectrometer (Ciphergen Biosystems, Go ¨ ttingen, Germany) was used to measure the molecular J. Reitz et al. Cholesterol binding of START proteins FEBS Journal 275 (2008) 1790–1802 ª 2008 The Authors Journal compilation ª 2008 FEBS 1799 [...]... Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol J Lipid Res 47, 2614–2630 30 Mathieu AP, Fleury A, Ducharme L, Lavigne P & LeHoux JG (2002) Insights into steroidogenic acute regulatory protein (StAR) -dependent cholesterol transfer in mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially... visualization system for exploratory research and analysis J Comput Chem 25, 1605–1612 1802 Supplementary material The following supplementary material is available online: Fig S1 Cholesterol binding of the START proteins Fig S2 Stability of the START domains of human STARD1 and STARD3 in the presence of photocholesterol or cholesterol This material is available as part of the online article from http://www.blackwell-synergy.com... 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JF III (1998) Steroidogenic acute regulatory protein (StAR) is a sterol transfer protein J Biol Chem 273, 26285–26288 10 Clark BJ, Wells J, King SR & Stocco DM (1994) The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells Characterization of the 1800 16 17 18 19 20 21 steroidogenic acute regulatory protein (StAR) J Biol... absence of its mitochondrial import sequence: implications for the mechanism of StAR action Proc Natl Acad Sci USA 93, 13731–13736 Arakane F, Kallen CB, Watari H, Foster JA, Sepuri NB, Pain D, Stayrook SE, Lewis M, Gerton GL & Strauss JF III (1998) The mechanism of action of steroidogenic acute regulatory protein (StAR) StAR acts on the outside of mitochondria to stimulate steroidogenesis J Biol Chem 273,.. .Cholesterol binding of START proteins J Reitz et al masses of polypeptides Typically, 1 lg of native protein (corresponding to 0.26 nmol of STARD1-START or 0.29 nmol of STARD3-START) or cleaved protein was added to one spot of H4-protein chips (reversed phase) Sinapinic acid or a-cyano-4-hydroxycinnamic acid (Ciphergen) were used as energy-absorbing matrices according to the manufacturer’s . Cholesterol interaction with the related steroidogenic acute regulatory lipid-transfer (START) domains of StAR (STARD1) and MLN64 (STARD3) Julian Reitz 1 , Katja. study addresses the question of whether or not the related START domains of StARD1 and StARD3 interact with cholesterol in a similar manner. Results Expression of the START domains The recombinant START. 35000 Intensity Intensity 0 5 10 15 20 0 10 20 30 40 50 29162.8+H 26167.8+H STARD1-START STARD3-START Fig. 1. Expression of the START domains of STARD1 and STARD3. (A) Domain organization of the START proteins STARD1 (285 amino acids) and STARD3 (445 amino