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 ß-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.

Quantum chemical investigations on intraresidue carbonyl-carbonyl contacts in aspartates of high-resolution protein structures.

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.

Self-contacts in Asx and Glx residues of high-resolution protein structures: role of local environment and tertiary interactions.

In protein structures, side-chains of asparagine and aspartic acid (Asx) and glutamine and glutamic acid (Glx) can approach their own backbone nitrogen or carbonyl group. We have systematically analyzed intra-residue contacts in Asx and Glx residues and their secondary structure preferences in two different datasets consisting of 500 and 1506 high-resolution structures. Intra-residue contact in an Asx/Glx residue between the heavy atoms of side-chain and main-chain functional groups of the same residue was investigated irrespective of whether such contacts are due to hydrogen bonding or not. Our search yielded 563 and 1462 cases of self-contacting Asx and Glx residues from the two datasets. Two important observations have been made in this analysis. First, self-contacts involving side-chain oxygen and backbone nitrogen atoms in majority of Asx residues are not due to hydrogen bonds. In the second instance, surprisingly, side-chain and backbone carbonyl oxygens of a significant number of Asx and Glx residues approach each other. For a wide-range of accessible surface areas, self-contacting residues are surrounded by less number of polar groups compared to all other Asx/Glx residues. In buried and partially buried regions, side-chain and main-chain functional groups of these residues together participate in simultaneous interactions with the available polar groups or water molecules. Asx/Glx residues with self-contacts are rarely observed in the middle of an alpha-helix or a beta-strand. Asx/Glx side-chain having contact with its own backbone nitrogen shows different capping preferences compared to those having contact with its backbone oxygen. Examples of proteins with multiple self-contacting Asx/Glx residues are found. We speculate that mutation of a self-contacting residue in the buried or partially buried region of a protein will destabilize the structure. The results of this analysis will help in engineering protein structures and site-directed mutagenesis experiments.