title: Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1.
abstract: Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.
authors: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors: S K
authors: SK
authors: Natarajan
authors: L J
authors: LJ
authors: Stern
authors: S
authors: S
authors: Sadegh-Nasseri
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism; D015374:Biosensing Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000235:genetics; D015798:HLA-DR1 Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D014443:Tyrosine / Q000235:genetics
chemical_list: D000596:Amino Acids; D000944:Antigens, Differentiation, B-Lymphocyte; D015798:HLA-DR1 Antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D000949:Histocompatibility Antigens Class II; D046911:Macromolecular Substances; D010446:Peptide Fragments; C086595:influenza hemagglutinin (306-318); C045084:invariant chain; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine; D005998:Glycine