Identification of the lateral interaction surfaces of human histocompatibility leukocyte antigen (HLA)-DM with HLA-DR1 by formation of tethered complexes that present enhanced HLA-DM catalysis.
Human histocompatibility leukocyte antigen (HLA)-DM is a major histocompatibility complex (MHC)-like protein that catalyzes exchange of antigenic peptides from MHC class II molecules. To investigate the molecular details of this catalysis we created four covalent complexes between HLA-DM and the MHC class II allele DR1. We introduced a disulfide bond between the naturally occurring cysteine beta46 on HLA-DM and an engineered cysteine on the end of a linker attached to either the NH(2)- or the COOH terminus of an antigenic peptide that is tightly bound on DR1. We find that when DM is attached to the NH(2) terminus of the peptide, it can, for all linker lengths tested, catalyze exchange of the peptide with a half-life a few minutes (compared with uncatalyzed t(1/2) > 100 h). This rate, which is several orders of magnitude greater than the one we obtain in solution assays using micromolar concentrations of HLA-DM, is dominated by a concentration independent factor, indicating an intramolecular catalytic interaction within the complex. A similar complex formed at the COOH terminus of the peptide shows no sign of DM-specific intramolecular catalysis. Restrictions on the possible interaction sites imposed by the length of the linkers indicate that the face of DR1 that accommodates the NH(2) terminus of the antigenic peptide interacts with the lateral face of HLA-DM that contains cysteine beta46.
Stratikos
Efstratios
E
Department of Cellular and Molecular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA. Stratikos@crystal.harvard.edu
Mosyak
Lidia
L
Zaller
Dennis M
DM
Wiley
Don C
DC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D006681:HLA-D Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015798:HLA-DR1 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D006681:HLA-D Antigens; C084245:HLA-DM antigens; D015798:HLA-DR1 Antigen; D046911:Macromolecular Substances; D009842:Oligopeptides; D011994:Recombinant Proteins; D003545:Cysteine