Enhancement of MICA affinity for NKG2D by combining multiple rational design strategies (58.17)

Abstract

We increased the affinity of MICA for the dominant immunoreceptor NKG2D by 50-fold using seven rationally designed mutations found through a stepwise process similar to affinity maturation. First we evaluated the stability of amino acid mutations in MICA located beneath the interface with NKG2D using RosettaDesign. These mutations were near a flexible region that folds when NKG2D binds. Sets of mutations predicted to mildly destabilize this region of MICA also increased its affinity for NKG2D as measured by surface plasmon resonance, with the best results from three mutations that increased affinity by 20-fold. The second design strategy optimized MICA residues that contact NKG2D. Ten single mutations proposed by RosettaDesign increased MICA-NKG2D affinity by 2-fold to 8-fold by SPR. Most combined surface mutants tested had subadditive effects on binding, but some enhanced binding through enthalpic stabilization. A combination of four of the five best single mutants increased affinity by 19-fold from wild-type. These sets of three and four mutations were combined in a MICA variant that bound NKG2D with 50 nM affinity. This variant in complex with NKG2D persisted through a size-exclusion column under conditions where the wild-type complex fell apart. These mutants were purified and tested in the context of undergraduate biochemistry courses in large quantities, so samples of purified recombinant high-affinity MICA are readily available for more tests.