Molecular dynamics of point mutated I-A(k) molecules expressed on lymphocytes

We have recently reported the lateral and rotational diffusion parameters f or I-A(kappa) molecules expressing various cytoplasmic truncations (Int. Im munol. 12 (2000) 1319). We now describe the membrane dynamics of I-A(kappa) with various mutations in the presumed contact region between alpha beta - heterodimers in an (alpha beta)(2) dimer of dimers structure. Such mutation s are known to strongly affect the antigen presentation ability of these mo lecules (Int. Immunol. 10 (1998) 1237-1249) but cause relatively small chan ges in the molecular dynamics of I-A(kappa). Lateral diffusion coefficients of I-A(kappa) wild-type molecules and mutants obtained via fringe fluoresc ence photobleaching recovery (FPR) ranged from 1.1 to 2.3 x 10-(10)cm(2)/s at room temperature while fractional mobilities averaged 75 +/- 6%. For all cell types examined, treatment with either hen egg lysozyme 46-61 peptide or db-cAMP reduced the I-A(kappa) mobile fraction by about 10% relative to untreated cells, suggesting that these treatments may increase lateral conf inement of class II in lipid rafts or cytoskeletal interactions of the mole cules. Wild-type I-A(kappa) and mutants capable of normal or partial antige n presentation exhibited, as a group, slightly longer rotational correlatio n times (RCT) at 4 degreesC than did mutants inactive in antigen presentati on, 14 +/- 4 versus 10 +/- 1 mus, respectively. Moreover, peptide, cAMP and anti-CD40 mAb treatment all increased rotational correlation times for ful ly- and partially-functional I-A(kappa) but not for non-functional molecule s. For example, 16 h peptide treatment yielded average RCTs of 28 +/- 12 an d 10 +/- 1 mus for the groups of functional and non-functional molecules, r espectively. Such modulation of the dynamics of functional class II molecul es is consistent with these treatments ' stabilization of class II or induc tion of new gene expression. Measurements of fluorescence resonant energy t ransfer between I-A(kappa), though complicated by cellular autofluorescence , averaged 6 +/- 7% over 15 cells or treatments: a result consistent with t he presence of a small fraction of I-A(kappa) as a dimer of dimers species. In summary, our results suggest subtle changes in the molecular motions of class II molecules correlate with a significant impact on class II functio n. Molecules active in antigen presentation exhibit more restricted motion in the membrane. and thus presumably more extensive intermolecular interact ions, than non-functional molecules. Further, treatments, such as db-cAMP a nd anti-CD40, which rescue antigen presentation by partially defective muta nts, appear to increase such interactions, several types of which have alre ady been reported for class II. A more detailed understanding of these phen omena will require both more sensitive biophysical tools and a more refined model of the role of class II intermolecular interactions in antigen prese ntation. (C) 2001 Elsevier Science B.V. All rights reserved.