Cholesterol lowering drug may influence cellular immune response by altering MHC II function.

Major histocompatibility complex class II (MHC II) expressed on the surface of antigen-presenting cells (APCs) displays peptides to CD4+ T cells. Depletion of membrane cholesterol from APCs by methyl β-cyclodextrin treatment compromises peptide-MHC II complex formation coupled with impaired binding of conformational antibody, which binds close to the peptide binding groove of MHC II. Interestingly, the total cell surface of MHC II remains unaltered. These defects can be corrected by restoring membrane cholesterol. In silico docking studies with a three-dimensional model showed the presence of a cholesterol binding site in the transmembrane domain of MHC II (TM-MHC-II). From the binding studies it was clear that cholesterol, indeed, interacts with the TM-MHC-II and alters its conformation. Mutation of cholesterol binding residues (F240, L243, and F246) in the TM-MHC-II decreased the affinity for cholesterol. Furthermore, transfection of CHO cells with full-length mutant MHC II, but not wild-type MHC II, failed to activate antigen-specific T cells coupled with decreased binding of conformation-specific antibodies. Thus, cholesterol-induced conformational change of TM-MHC-II may allosterically modulate the peptide binding groove of MHC II leading to T cell activation.


Ethics statement
Use of mice was approved by the Institutional Animal Ethics Committee of the Indian Institute of Chemical Biology, India. All animal experimentations were performed according to the National Regulatory Guidelines issued by the Committee for the Purpose of Supervision of Experiments on Animals (CPSEA), Ministry of Environment and Forest, Government of India.

Monoclonal antibodies
The following antibodies were used: AMS32.1 (IgG2b , reacts with I-A of d, f, g7, i, and v haplotypes); 11.5-2 (IgG2b , reacts with I-A of k and r haplotypes), 10-2.16 (IgG2b , reacts with I-A of k, r, f, and s haplotypes). The m2C44 cell line, specifi cally recognized LACK 156

Isolation of peritoneal exudate cells
BALB/C and CBA/J mice (8-10 weeks old) were intraperitoneally injected with 3 ml of 4% starch. After 48 h, peritoneal exudate cells (PECs) were isolated and plated on tissue culture petri dishes (1 × 10 6 cells/ml) or glass cover slips (1 × 10 5 cells/ml), in 10 ml or 0.5 ml, respectively, of complete RPMI medium for 48 h at 37°C in a humidifi ed 5% CO 2 incubator . Nonadherent cells were removed thereafter by gentle washing with serum-free medium. The adherent PECs are defi ned as macrophages (M ⌽ s) for convenience henceforth.

m ␤ -CD treatment and liposomal treatment
The M ⌽ s were treated with 10 mM m ␤ -CD in RPMI for 20 min in order to scavenge membrane cholesterol as described by others ( 10 ). m ␤ -CD was dissolved in RPMI 1640. Liposomal cholesterol was prepared with cholesterol and phosphatidylcholine (PC) at a molar ratio of 1.5:1 as previously described ( 12 ). Similarly, liposomal cholesterol analog was prepared cholesterol analog (4-cholestene-3-one) and PC as described ( 12 ). To alter the fl uidity of cells, 10 5 cells/100 l were incubated with 10 l liposomes for 4 h at 37°C in RPMI ( 10 ). The cells were fi xed with 4% paraformaldehyde for 10 min and followed by washing three times in serum-free RPMI.

Flow cytometry
The cell surface binding of conformation-specifi c antibodies was measured by FITC-labeled anti-mouse A k (clone 11-5.2). The total cell surface expressions of A k were measured by anti-mouse A k (clone 10-2.16 or clone 10-3.6). mAb 10-2.16 and mAb 10-3.6 recognized the same epitope Ia.17 (we have used these two mAbs interchangeably) ( 27 ). The cells were stained with goat antimouse FITC while using mAb 10-2.16 in PBS containing 5% FBS . The mean fl uorescence intensity (MFI) for FITC was determined in a FACSAria II system (BD Bioscience, San Diego, CA).  (20 M) or HEL 46-61 peptide (20 M), respectively. Briefl y, 5 × 10 5 T hybridoma cells were cocultured with 1 × 10 5 appropriate M ⌽ s in the presence of respective antigen. The M ⌽ s used in this study were CCR5 is altered due to cholesterol removal. Previously we showed that in Leishmania donovani infection there is a signifi cant decrease in membrane cholesterol ( 12 ) and serum cholesterol ( 13 ) coupled with defective T cell stimulating ability ( 14 ), and impaired IFN-␥ receptor subunit assembly ( 15 ). The above defect could be corrected by liposomal cholesterol ( 14,15 ).

Stimulation of T cell hybridoma upon m ␤ -CD treatment of APCs
Structure activity analysis shows that cholesterol's effects are due to specifi c sterol-protein interactions, as shown in the case of a number of membrane bound receptors, such as those for cholecystokinin (type B), oxytocin, and nicotinic acetylcholine ( 16 ). Refi ned structure of the nicotinic acetylcholine receptor has been shown to have internal sites capable of forming adducts with cholesterol and resulting in stabilization of the protein structure ( 17 ). Both oxytocin and serotonin 1A receptors contain the strict cholesterol consensus motif (CCM), and in both there is a dramatic increase in agonist affi nity in the presence of cholesterol ( 18,19 ).
It is well known that MHC II can adopt multiple conformations with distinct activities ( 20,21 ). The conformational changes of MHC II during biosynthesis, folding, and in the MHC class II-containing compartment were detected by monoclonal antibody (mAb ) binding (22)(23)(24)(25). The subtle conformational changes of MHC II upon binding of peptide were detected by mAb binding ( 26 ). Thus conformational antibody is a powerful tool to study the conformational change of MHC II. The Ia.2 epitope is a lipid raft-associated conformer of MHC II, which is essential for B cell-T cell interaction. Binding of anti-Ia.2 mAb, such as 11-5.2, is highly dependent on the residues arginine-57 and glutamine-75 of the I-A k ␣ chain, residues near to the peptide binding groove ( 27 ). Thus it may be possible that membrane cholesterol may play an important role in maintaining the active form of MHC II.
Our study shows, for the fi rst time, that depletion of membrane cholesterol from APCs reduces peptide-MHC II complex formation and also binding of conformationspecifi c mAb 11-5.2, but not the nonconformational mAb. Interestingly enough, the transmembrane domain of MHC II (TM-MHC-II) interacts with cholesterol with high degree of specifi city leading to changes in the conformation of the transmembrane (TM) domain. Transfection of CHO cells with full-length mutant MHC II showed reduced T cell stimulating ability and binding of conformation-specifi c mAb 11-5.2 as compared with wild-type MHC II. Thus membrane cholesterol plays an important role in maintaining the active form of MHC II.
(stock in ethanol) to the mixture of NBD-cholesterol and TM-MHC-II.

Side-directed mutagenesis and transfection
PCR QuickChange mutagenesis kit (Stratagene) was used to make amino acid substitutions at positions F240, L243, and F246 in the ␤ chain of A k (A k plasmid was kind gift from Jim Drake, Albany Medical College). The amino acids were replaced with alanine. Mutagenesis was confi rmed by DNA sequencing. The plasmids used for transfection were wild-type A k (A k ) and mutant A k . CHO cells were grown in a 60 mm petri-dish with 2 × 10 5 cells. The cells were allowed to adhere for 16 h and nonadherent cells were removed by washing. The transfections were performed with 2 g of each plasmid using lipofectamine according to manufacturer's protocol ( 15 ). Then the cells were kept in resting condition for 24 h in RPMI with 10% FBS .

T cell stimulation depends on membrane cholesterol but not on membrane fl uidity
Cholesterol regulates membrane fl uidity of eukaryotic cells ( 16 ). Our study showed that treatment of M ⌽ s with showed compromised ability to stimulate T cell hybridoma (HyH12.6, A k restricted) as evident from the 80% decrease in IL-2 production as compared with N-M ⌽ s in presence of HEL 46-61 peptide, whereas m ␤ -M ⌽ -CL showed normal T cell stimulating ability ( Fig. 1C ). To show that the observed effect was not unique to T cell hybridoma HyH12.6, we used another T cell hybridoma (LMR7.5, A d restricted). In this context LMR7.5-stimulating ability of BALB/Cderived M ⌽ s was assayed in the presence of LACK 156-173 peptide. m ␤ -M ⌽ s and m ␤ -M ⌽ -CL-AN showed 70% decreased IL-2 production in the presence of LMR7.5 as compared with N-M ⌽ s and m ␤ -M ⌽ -CL ( Fig. 1D ). This observation clearly indicates that there is a specifi c need of cholesterol, and restoration of membrane fl uidity of APCs is not enough to present MHC II-restricted antigens to T cells.

Peptide-MHC II complex formation depends on membrane cholesterol
Because peptide-MHC II complex formation is the initial event of T cell activation, we studied peptide-MHC II fi xed with 4% paraformaldehyde. Both T cells and M ⌽ s were kept for 24 h in complete medium at 37°C in humidifi ed 5% CO 2 . The resulting culture supernatant was assayed for IL-2 by ELISA.

Homology modeling of MHC II protein and cholesterol docking
Primary sequences of ␣ (UniProt ID: P04228) and ␤ chains (UniProt ID: P01921) of MHC II protein were supplied to PH-Dhtm, DAS, and TMHMM servers for the prediction of the TM regions. Consensus predicted TM helices were 26 (region: T219-L244) and 21 (region: M227-I247) residues in length for chains ␣ and ␤ , respectively. The sequence encompassing the TM helices and the fl anking loop regions were subjected to the HHPred fold recognition server. Specifi c chains of receptor tyrosine kinases, EphA2 (PDB ID : 2K9Y, chain A) and ErbB2 (PDB ID: 2JWA, chain A) were used as templates to build the three-dimensional (3D) models of the predicted TM helix regions in chains ␣ and ␤ . Suitable 3D models of TM helices and the cytoplasmic loops were generated using the Modeler v9.9 package and fi ltered based on the best energy parameters (MOLPDF and DOPE scores) followed by critical visual inspection. Side chains were rebuilt using the SCWRL 4.0 program. The fi nal models were utilized to identify the probable ␣ -␤ TM helix dimerization and packing using PATCHDOCK ( 28 ) docking server followed by vacuum molecular dynamics simulation using GROMACS v4.5.3 ( 29 ) molecular modeling package. Using CHIMERA v1.5.3 software, best orientation of dimeric TM helices along with the cytoplasmic loops were brought in the same reference frame of the X-ray coordinates of A d extracellular domain structure [PDB ID: 2IAD]. Further, Modeler v9.9 was used to build the suitable loops connecting the extracellular domains to the TM-MHC-II. The stereochemical properties of the fi nal assembled model were validated by PROCHECK, WhatCheck, Verify_3D, and Errat programs (supplementary Table II). PATCHDOCK server was availed to fi nd probable docking solutions of cholesterol binding to the MHC II model. For more details on the homology modeling see supplementary data.

Analysis of binding of cholesterol to TM-MHC-II
All fl uorescence studies were done in a Perkin Elmer LS55 spectrofl uorometer at 25°C and the experiments were carried out in a 1 cm path length quartz cuvette. NBD-cholesterol was used to monitor binding of cholesterol with TM-MHC-II. Binding of TM-MHC-II (25 M stock in TFE) and mutant TM-MHC-II to cholesterol was measured by addition of increasing concentration of the NBD-cholesterol to 10 nM peptides in PBS (pH 7.2) containing 2 mM CHAPS. Similarly, increasing concentration of NBD-cholesterol was added to PBS and the 10 nM concentration of peptide added to PBS as a control. NBD-cholesterol was excited at 470 nm and emission measured from 500 to 570 nm ( 30,31 ). The buffer controls were subtracted from fl uorescence data. The steady state NBD-cholesterol binding was analyzed using a hyperbolic equation (one binding site) by plotting the ratio of fl uorescence intensity versus the NBD-cholesterol concentration.
where C is the concentration of NBD-cholesterol, F i is the fl uorescence intensity at 520 nm at C, F max is the fl uorescence intensity NBD-cholesterol at 400 nm NBD-cholesterol, and K d is the dissociation constant of the binding. The binding of NBD-cholesterol to TM-MHC-II was measured also in PBS with 2 mM PC and PBS containing 30% TFE.
Specifi city of the binding was determined by incubating 100, 600, and 1,100 nM of unlabeled cholesterol and analog cholesterol complex formation in m ␤ -M ⌽ s and also upon liposomal treatments. The peptide-MHC II complex formation was measured by direct demonstration of the LACK 156-173 -A d complex on the cell surface using LACK 156-173 -A d complexspecifi c mAb (m2C44) binding followed by staining with FITC-labeled goat anti-mouse IgG. The formation of peptide-MHC II complex was expressed in terms of MFI. It was observed that m ␤ -M ⌽ s and m ␤ -M ⌽ -CL-AN showed reduced peptide-MHC II complex as compared with N-M ⌽ s. m ␤ -M ⌽ -CL showed increased peptide-MHC II complex, which is comparable to N-M ⌽ s ( Fig. 2 ).

Reducing membrane cholesterol alters the binding of conformation-specifi c antibodies directed toward MHC II protein
Binding of conformation-specifi c antibodies to MHC II was monitored by FITC-labeled mAb 11-5.2, which specifically recognizes the Ia.2 epitope. mAb 11-5.2 binds close to the peptide binding groove, and arginine-57 and glutamine-75 are critical for the binding ( 27 ). It was observed that m ␤ -M ⌽ s showed decreased binding of mAb 11-5.2 by 4-fold, whereas the total cell surface of MHC II remained unaltered as evident from binding of nonconformationspecifi c mAb (10-2.16). The treatment of m ␤ -M ⌽ s with liposomal cholesterol increased the binding of mAb 11-5.2, but not by liposomal cholesterol analog treatment ( Fig. 3 ).

Cholesterol binding motif in MHC II
A careful search of the amino acid sequences within the membrane spanning regions of MHC II protein revealed substantial resemblance to cholesterol recognition/interaction amino-acid consensus (CRAC)-like ( 32 ) and CCMlike ( 33 ) motifs . The putative CRAC-like motif was located at the C-terminal region of the TM-MHC-II in the ␣ chain [V 234 -(X) 4 -F 239 -(X) 5    (TM-MHC-II) was synthesized by using quasi-orthogonally protected Lys as linker amino acid ( Fig. 4B ). To increase the solubility of the peptide, six Lys residues were added at the N-terminal end (M227) of the ␤ chain ( 35,36 ).

Cholesterol changes conformation of TM-MHC-II
Binding of cholesterol with TM-MHC-II was monitored by the change in fl uorescence spectrum of NBD-cholesterol ( 30,31 ). This revealed that the fl uorescence emission spectrum of NBD-cholesterol undergoes a large increase in intensity in the presence of TM-MHC-II in PBS containing 2 mM CHAPS (detergent) ( Fig. 5A ), together with a blue shift of 40 nm in em from 560 to 520 nm, indicating that the sterol was located in a hydrophobic pocket when bound to the peptide. The increasing concentration of A better understanding of the cholesterol-mediated modulation of the peptide-MHC II complex formation requires MHC II 3D structure at atomic details. As membrane proteins pose a lot of experimental challenges to decipher data for a high resolution structure ( 34 ), we circumvented the diffi culty by building a reliable heterodimer model for MHC II ␣ and ␤ chains using a careful combination of fragment model assembly and molecular dynamics approaches (see Materials and Methods) (supplementary Fig. II). In our model, the TM-MHC-II is predominantly composed of nonpolar residues like Gly, Val, Leu, and Ile from ␣ chains (residues T219-L244) and ␤ chains (residues M227-I247). Highly conserved GxxxG motifs, G 225 -(X) 3 -G 229 and G 232 -(X) 3 -G 236 in the ␣ chain and G 233 -(X) 3 -G 237 in the ␤ chain, formed the interface of the helix-helix dimer in our 3D model. The fi rst three Gly residues of the GxxxG motif from the ␣ chain (G225, G229, and G232) in this model are found to be within 4 Å mutual distances from G233: ␤ and G237: ␤ , thus assisting in close packing of the TM-MHC-II ( Fig. 4A ). G230: ␤ and G244: ␤ , which are not part of any GxxxG motif but are closely placed to G225: ␣ and G236: ␣ , may also assist in helix-helix dimerization. The docking of cholesterol to MHC II predicted more than one binding site based on surface complementarities, but a majority ( ‫ف‬ 56%) of the binding was restricted to the lower half of TM-MHC-II. The docking solutions obtained using PATCHDOCK ( 28 ) demonstrated probable binding regions of cholesterol within the TM-MHC-II, which was close to the predicted CRAC/CCM-like motif ( Fig. 4A ). The interacting distances between the cholesterol molecules and the residues encompassing the region of CRAC-or CCM-like motifs are shown in supplementary Table I.
Using the homology model of the TM helices as a reference where the C ␣ -atoms of the C-terminal residues L244: ␣ and I247: ␤ are ‫ف‬ 8.5 Å apart, the helices are designed in a manner so that an Ala-Lys linker could maintain a similar distance. The heterodimeric branched peptide of MHC II   ( Fig. 5C ). The K d for mutant TM-MHC-II was 1,002 nM and 1,119 nM in the presence of PC and 30% TFE, respectively ( Table 1 ). Mutation of cholesterol binding residues reduces the K d ‫ف‬ 10-to 20-fold, as compared with TM-MHC-II.
Cholesterol-dependent conformational change of TM-MHC-II was measured by circular dichroism. The helical content was calculated using the mean residual ellipticity was measured at 222 nm ( 37 ). The helical content of TM-MHC-II in PC liposome (containing only PC) and cholesterol liposome (PC:cholesterol, 1:1) was 84 and 68%, respectively ( Fig. 5D ). We further verifi ed the conformational change of TM-MHC-II. As the conformation of TM peptide varies in different solvents ( 38 ) and TFE can mimic the membranelike environment ( 39 ), we studied the secondary structure of NBD-cholesterol increased fl uorescence intensity, which then reached a plateau; the K d value for TM-MHC-II was 31 nM in presence of CHAPS ( Fig. 5B ). We have validated binding of cholesterol with TM-MHC-II using lipid (PC) and membrane-mimicking solvent (TFE). It was observed that the K d value for TM-MHC-II was 77 nM and 50 nM in the presence of PC and 30% TFE, respectively ( Table 1 ).
The specifi city of the binding of NBD-cholesterol to TM-MHC-II was determined by competition experiments with unlabeled cholesterol and also by mutant TM-MHC-II. The residues F240, L243, and F246 in the TM-MHC-II are predominantly hydrophobic and may favor van der Waal's interaction with the cholesterol molecule (supplementary Table I  TM-MHC-II in the presence of 30% TFE. The helical content of TM-MHC-II with and without cholesterol was 63 and 79%, respectively ( Table 2 ).

Mutation of cholesterol binding residues of MHC II alter the binding of conformation-specifi c antibody and T cell stimulating ability
To study the importance of cholesterol binding at the appropriate motif on the distal peptide binding groove of MHC II, we mutated putative cholesterol binding residues (F240, L243, and F246 of the ␤ chain) in the TM domain of full-length MHC II (mutant A k ) and its effects on the nature of the peptide binding groove was monitored using binding of conformation-specifi c mAb and T cell stimulating ability. CHO cells were transfected with either A k or mutant A k , which were used as APCs. The binding of conformation-specifi c antibody was assessed using mAb 11-5.2 and total cell surface expression was assessed using nonconformational antibody (10-3.6). The resulting binding was expressed in terms of MFI. Our study showed that the cell surface expression of MHC II remains unaltered in mutant A k whereas binding of conformational antibody was reduced by ‫ف‬ 5-fold as compared with CHO cells expressing wild-type MHC II ( Fig. 6A ). The ability of transfected cells to stimulate T cells was assayed in terms of IL-2 production in the presence of HEL 46-61 peptide. It was observed that mutant A k showed compromised ability to stimulate T cell hybridoma (HyH12.6, A k restricted) as evident from the decreased IL-2 production as compared with wild-type A k ( Fig. 6B ).

DISCUSSION
Our study very clearly showed that decrease in membrane cholesterol not only compromised peptide-MHC II stability but also compromised binding of conformationspecifi c mAb (11-5.2) directed toward MHC II protein ( Figs. 2, 3 ). This decreased binding of conformationspecifi c antibodies is not due to reduced total cell surface expression of MHC II because binding of nonconformationspecifi c antibodies remains unaltered ( Fig. 3 ). The treatment of m ␤ -M ⌽ s with liposomal cholesterol largely restored  11-5.2. The binding of mAb 10-3.6 (nonconformational antibody) to A k and mutant-A k was to assess total cell surface expression of A k (open box). The cells were stained with PE -conjugated mAb 10-3.6. The binding was expressed in terms of relative MFI with respect to A k . MFI of A k transfected cells were considered as 1,000. B: The anti-HEL T cell hybridomas (HyH12.6) were cocultured with A k and mutant-A k in the presence of HEL 46-61 peptide (100 M). The resulting IL-2 productions in the supernatant were measured by ELISA. *** P < 0.0005 with respect to N-M ⌽ s. membrane cholesterol ( Fig. 1A ), and this is in support of the observation by others ( 10 ). There is a report that quenching of membrane cholesterol disrupts lipid raft and replenishment of membrane-restored lipid raft formation ( 40 ). The loading of membrane cholesterol restored membrane rigidity, peptide-MHC II complex formation, and binding of conformation-specifi c mAb ( Figs. 1-3 ). The effect of cholesterol may be due to specifi c interaction between MHC II and cholesterol or due to an increase in the membrane rigidity. It has been shown that function of the cholecystokinin receptor is related to the membrane fl uidity ( 41 ), whereas function of the oxytocin receptor is not related to membrane fl uidity but there is a specifi c requirement of cholesterol ( 16 ). We showed that liposomal cholesterol analog (4-cholestene-3-one) treatment increased the membrane rigidity but failed to restore peptide-MHC II complex formation and binding of conformation-specifi c mAbs ( Figs. 1-3 ), suggesting the specifi c role of cholesterol. Thus, the peptide-MHC II complex formation and binding of conformation-specifi c mAbs depends on membrane cholesterol content. The role of membrane cholesterol in fi ne tuning of membrane proteins is of great interest. X-ray crystallographic structures give direct evidence of cholesterol physically bound to membrane proteins, e.g., ␤ 2 -adrenergic receptor (PDB ID: 3D4S) ( 33 ), proton pumping rhodopsin (PDB ID: 3AM6) ( 42 ), sodium-potassium pump (PDB ID: 2ZXE) ( 43 ), etc. To show any possible interaction between MHC II and cholesterol we endeavored to build a of cholesterol in the TM domain of MHC II may change the conformation of the peptide binding groove in an allosteric pathway and the conformational change of the peptide binding groove may be either by specifi c pathway or by global conformational change of MHC II. Many human diseases, such as leishmaniasis and tuberculosis, are associated with reduced cholesterol level ( 13,50 ) coupled with immunosuppression ( 14,51 ). It was observed that under certain pathological conditions, like leishmaniasis, there is an expansion of CD8 + T cells coupled with a decline of CD4 + T cells ( 52,53 ). It has been shown that statins, cholesterol lowering drugs, reduce IFN-␥ -inducible expression of MHC II, but do not reduce constitutive expression of MHC II in professional APCs ( 54 ). High density lipoprotein (HDL) is an acceptor of cholesterol ( 6 ) and HDL treatment reduces MHC II-restricted T cell activation ( 55 ). It has been shown that statin treatment increases HDL level in the plasma ( 56 ), thus statin-induced cholesterol effl ux may be mediated by HDL or by an HDL independent event. There is a report that thymic development of immune repertoire is dependent on cholesterol. The disruption of the lipid raft of thymic epithelial cells leads to impaired CD4 + T cell expansion, but there is enhanced CD8 + T cell expansion because of lipid raft association of MHC II but not MHC I ( 3 ). This observation also supports the notion that cholesterol depletion impairs MHC II-restricted, but not MHC I-restricted, T cell stimulation. The role of membrane cholesterol in APCs has been well studied in various systems ( 4,41,57,58 ). Changes in fl uidity are reported to have strong bearing in T cellmediated cytotoxicity ( 57,59,60 ) and NK cell-mediated target cell lysis ( 61 ). Thus cholesterol may play a decisive role in governing immune response of the host. This raises the issue that long term therapy with cholesterol lowering drugs may induce immunosuppression. In summary, we tend to believe that membrane cholesterol interacts with MHC II protein. Such interaction may be important to maintain the peptide binding groove in an active form to bind peptide and subsequently activate T cells. Depletion of membrane cholesterol by pharmacological mediators may infl uence the MHC II-restricted immune repertoire, the outcome of which may be generalized immunosuppression.
reliable 3D model of full-length MHC II, and molecular docking analysis suggests probable cholesterol binding residues are located at the C-terminal regions of the TM domain from the ␣ and ␤ chains ( Fig. 4 ). The affi nity of cholesterol was stronger for the TM domain of the ␤ chain than that of the ␣ chain (unpublished observation), therefore, we have mutated (F240, L243, and F246) probable cholesterol binding residues in the ␤ chain of TM-MHC-II (mutant TM-MHC-II). Our study very clearly showed that the binding of cholesterol with TM-MHC-II was specifi c because of a dramatic decrease (10-to 20fold) in K d in mutant TM-MHC-II ( Fig. 5C ), and the binding of NBD-cholesterol could be competed out faster by unlabeled cholesterol as compared with cholesterol analog (supplementary Fig. III). Most interestingly, there was a signifi cant change in the secondary structure of TM-MHC-II in the presence of cholesterol ( Fig. 5D ). Mutation of cholesterol binding residues (F240, L243, and F246) in the full-length MHC II (mutant MHC II) and subsequent transfection of CHO cells with mutant MHC II showed decreased binding of conformation-specifi c mAbs, but interestingly, cell surface MHC II remained unaltered. It was observed that CHO cells expressing mutant MHC II showed ‫ف‬ 3-fold less T cell stimulation as compared with CHO cells expressing wild-type MHC II ( Fig. 6 ).
It may be recalled that binding of conformation-specifi c antibody is a reliable tool to study conformational change of MHC II (22)(23)(24)(25)(26). Thus, it is likely that in the absence of cholesterol, conformation of the peptide binding groove may have changed with decreased binding of immunogenic peptide to the peptide binding groove of MHC II protein. Naturally one can ask how changes in the conformation of TM-MHC-II in the presence of cholesterol distally infl uence the conformation of the peptide binding groove of MHC II. Molecular dynamic study demonstrated that cholesterol interacts with rhodopsin to induce local structural perturbation, which in turn brings global conformational rearrangements through rigid body motions ( 44 ). Cholesterol-dependent cytolysins are families of pore-forming toxins which contain a highly conserved undecapeptide motif, which is essential for cholesterol-mediated membrane binding. The membrane binding with this undecapeptide leads to structural change in the distal region of cholesterol-dependent cytolysins in an allosoteric pathway and the structural change is necessary for the monomer-monomer interaction that drives assembly of the ␤ -barrel pore ( 45 ). Binding of ligands in the extracellular domain of GPCR, an integral membrane protein, dynamically changes its conformation in an allosteric pathway that is refl ected in the cytosolic domain which interacts with G protein ( 46 ). Molecular dynamic study showed that the membrane proximal domain of MHC II has profound infl uence on the peptide binding groove ( 47 ). Allosteric modulator changes the conformation of the binding site either by specifi c pathways that link the allosteric site with the binding site ( 48 ) or by global conformational change of the molecule, which in turn affect the conformation of the binding site ( 49 ). Thus, it can be concluded that binding