MUTATIONAL ANALYSIS OF 2 DR-ALPHA RESIDUES INVOLVED IN DIMERS OF HLA-DR MOLECULES

Crystallographic analysis of HLA-DR1 molecules reveals a ''dimer of di mers'' with two reciprocal salt bridges between Glu 88 and Lys 111 of the two DR alpha chains. To determine whether these amino acids are cr itical for Ag presentation, we generated a panel of human B cell trans fectants expressing DR alpha chains with mutations at residues 88, 111 , or both. The mutant DR alpha chains, paired with endogenous DR3 beta chain, form cell surface dimers that retain epitopes recognized by a panel of anti-DR3 Abs. Replacement of Glu 88 with Ala (88A) selectivel y eliminates the ability to activate an alloreactive (anti-DR3) T cell clone. Mutant DR molecules with Lys substituted for Glu 88 (88K) fail to activate an alloreactive, an Ag-specific, and a peptide-specific T cell line. The DR alpha 88 mutants bind an exogenously supplied DR3-s pecific peptide and the mutant DR molecules migrate as dimers on SDS-P AGE, implying that their defective Ag presentation is not due to an in ability to bind antigenic peptides. In contrast, substitution of Lys 1 11 with either Ala (111A) or Glu (111E) does not abrogate Ag presentat ion. Further, the defect introduced by Glu 88 to Lys mutation (88K) is not overcome by compensatory Lys to Glu mutation at position 111 (111 E). Taken together, these results indicate an important functional or structural role for position 88 of the DR alpha chain, but argue again st a requirement for interaction between DR alpha 88 and 111 during Ag -specific T cell stimulation.