Structural analysis of the interaction of apamin with Ia and its recognition by Ad- or Ab-restricted mouse T cells.

Apamin is a single-chain, disulfide-bonded, 18-amino acid peptide that elicits mouse T cell responses when presented by cells expressing syngeneic Ad or Ab class II MHC molecules. We previously showed that both the unfolding of this peptide by APC and the integrity of its N terminus segment were required for efficient apamin T cell recognition. To seek further information on the sites through which this peptide interacts with Ia and/or TCR, we used a panel of Ad- or Ab-restricted, apamin-specific THC to probe the antigenicity of a series of synthetic apamin analogs. These included peptides either truncated at the N terminus, or substituted by Ala at position 2, 4, 6, 7, 8, or 10. Analysis of THC responses to apamin analogs and use of the latter in competition assays for peptide presentation revealed the following: 1) optimal apamin T cell recognition critically involved Lys4, Ala5, Pro6, Glu7, and Leu10. The role of these residues in either "Ia or TCR binding regions" was found to depend upon the restricting Ia molecules at play. Thus, Lys4, Glu7, and Leu10 were TCR-binding residues in both Ad- and Ab-apamin complexes, whereas Lys4 participated in apamin/Ab but not, or to a marginal extent, in apamin/Ad interaction. Furthermore, Pro6 was associated either with an Ia contact region or a TCR interaction site when apamin was presented by Ab or Ad molecules, respectively. Unfolded apamin and the unrelated chicken OVA323-339 peptide were found to bind to the same, or closely related site(s) of Ad, as shown by their ability to compete reciprocally for recognition by appropriate Ad-restricted THC. Four distinct TCR V beta genes (V beta 2, V beta 4, V beta 6, and V beta 8) were found to be used in our panel of 16 apamin-specific THC. These data indicate that apamin interacts with Ad or TCR through a motif resembling other beta-sheeted, Ad-binding sequences; however, based on the spacing of the critical residues (i.e., 4, 7, and 10), the possibility exists that apamin processing permits the folding of this sequence into an alpha-helix.