Identification of essential residues of CTLA-2α for inhibitory potency

To identify functionally essential sequences and residues of CTLA-2α, in vitro mutagenesis was carried out. The coefficient of inhibition (K i ) was determined towards rabbit cathepsin L using Z-Phe-Arg-MCA as the substrate. Recombinant CTLA-2α inhibited the enzyme potently (K i = 15 nM). A truncated mutant, lacking the N- and C-terminal Ala1-Asp9 and Leu80-Glu109 regions, was also a potent inhibitor (K i = 10 nM). Subsequent short deletions in the central region (Asn10-Ser79) showed three functionally essential distinct regions: Asn10-Phe19, His30-Ala44 and Ser55-Ser79. These regions cover sequences corresponding to three helices (α1, α2 and α3) and sequences that interact with the cognate enzyme. Alanine scanning showed that replacement of one of three conserved Trp residues increased the K i by 15-20-fold; whereas, replacement of two/three Trp residues at once caused complete loss of potency, as did replacing Cys75 with Ala or Ser. The proteins from wild-type (WT) CTLA-2α and mutant C75A were stable overnight when incubated with cathepsin L; whereas, proteins from mutants W12A, W15A and W35A were quickly digested. Incubation of cathepsin L/WT CTLA-2α formed a complex; whereas, C75S did not form a complex. Our overall results point to a critical role of W12, W15, W35 and Cys75 residues in CTLA-2α.