There is a report that lowering of membrane cholesterol decreases expansion of the immune repertoire of CD4 + T cells, but not CD8 + T cells, in the thymic organ culture ( 3 ). The association of MHC II with either lipid raft or tetraspanin membrane domain is essential for effective antigen presentation ( 4,5 ). The treatment of antigen-presenting cells (APCs) with methyl ␤ -cyclodextrin (m ␤ -CD), known to deplete cellular cholesterol ( 6 ), reduces the antigen-presenting ability of MHC II without altering cell surface expression of MHC II ( 4 ). MHC II-restricted cognate interaction between APCs and CD4 + T cells is necessary for the initiation and propagation of immune response ( 7 ). Collectively the above information inclined to indicate that membrane cholesterol may play a decisive role in the expansion and maintenance of the immune repertoire, but the mechanism is largely unknown. This work is designed to understand how membrane cholesterol infl uences immune response.Cholesterol is important for lipid raft assembly ( 8 ) and also important for the formation of a tetraspanin-enriched microdomain ( 9 ). At a low cholesterol concentration, the diffusion coeffi cient of GPI-linked MHC II is reduced by a factor of 190 ( 10 ). There is a report that cholesterol depletion from the cell decreases the binding of anti-CCR5 antibodies directed to different sites of the receptor in a varying degree ( 11 ) The cholesterol lowering drug, statin, is extensively used in medical practice. There are clinical reports suggesting a better outcome of cardiac transplant in patients on statin therapy ( 1 ). Statin inhibits IFN-␥ -induced major histocompatibility complex class II (MHC II) expression but does not affect constitutive expression of MHC I and MHC II ( 2 ). Research, New Delhi, India and Network Project (Project NWP 0005 / BSC 0120 ; APC, antigen-presenting cell; CCM, cholesterol consensus motif; CRAC, cholesterol recognition/interaction amino-acid consensus; 3D, three-dimensional; m ␤ -CD, methyl ␤ -cyclodextrin; m ␤ -M ⌽ , methyl ␤ -cyclodextrin-treated macrophage; m ␤ -M ⌽ -CL, methyl ␤ -cyclodextrintreated macrophages treated with liposomal cholesterol; m ␤ -M ⌽ -CL-AN, methyl ␤ -cyclodextrin-treated macrophages treated with liposomal cholesterol analog; M ⌽ , macrophage; MHC I/II, major histocompatibility complex class I/II; mAb, monoclonal antibody; MFI, mean fl uorescence intensity; N-M ⌽ , normal macrophage; N-M ⌽ -CL, normal macrophages treated with liposomal cholesterol; PC, phosphatidylcholine; PEC, peritoneal exudate cell; TFE, trifl uoroethanol; TM, transmembrane; TM-MHC-II, transmembrane domain of major histocompatibility complex class II. This work was supported by the Council of Scientifi c and Industrial

The folding and stability of a polypeptide chain are due to many different and simultaneous noncovalent interactions. Recent studies have observed several novel and counterintuitive contacts in protein structures, and the nature of interactions due to such contacts is yet to be fully elucidated. We have identified carbonyl-carbonyl intraresidue contacts in 102 Asp residues from a data set of high-resolution protein structures. At the outset, it appears that such close approach of two carbonyl oxygen atoms is energetically not favorable. We have carried out ab initio quantum chemical calculations on 10 representative examples of self-contacting Asp residues from different regions of the Ramachandran map. Potential energy scan using three levels of theory (HF, B3LYP, and MP2) and two basis sets (6-31+G* and 6-31++G**) was performed by varying the side-chain dihedral angle chi(1) while keeping all other parameters corresponding to that observed in the protein structures. We also calculated interaction energies by considering the surrounding interacting residues and water molecules. Our results show that the energy difference between a self-contacting Asp residue from the crystal structures and the minimum energy conformations is about 10-15 kcal/mol. This small energy difference is compensated by its interactions with the surrounding residues and water molecules as observed in the interaction energy analysis. The results are independent of the levels of theory used. The contacting carbonyl-carbonyl groups adopt a sheared parallel motif orientation which helps to expose both the backbone and side-chain carbonyl oxygen atoms and enable them to participate in tertiary interactions. Natural bond orbital calculations indicate that carbonyl-carbonyl groups in self-contacting Asp residues interact through n --> pi* electron delocalization. The geometry analysis and nature of chemical interactions together explain the rationale for the existence of such Asp residues in protein structures and their importance in the protein stability.

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