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*
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
Natarajan, S.K., M. Assadi and S. Sadegh-Nasseri. Stable peptide binding to MHC class II molecule is rapid and is determined by a receptive conformation shaped by prior association iwth low-affinity peptides. J. Immunol. In press.
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 ßGly86 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 
/ß 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.
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