SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef

The avid binding of HIV-1 Nef to the Src homology-3 (SH3) domain of Hck (K- D 250 nM) has been shown to involve an interaction between the RT-loop of H ck-SH3 and residues in Nef outside of its prototypic polyproline type II (P PII) helix-containing SH3-ligand region. Such distinctive interactions are thought to provide specificity and affinity for other SH3/ Ligand protein c omplexes as well. Here, we have constructed and successfully displayed on t he surface of M13 bacteriophage particles a complex library of SH3 domains, which are derived from Hck but carry a random hexapeptide substitution in their RT-loops (termed RRT-SH3). Using this strategy we have identified ind ividual RRT-SH3 domains that can bind to Nef up to 40-fold more avidly than Hck-SH3. Some of these high-affinity RRT-SH3 domains resembled Hck-SH3 in that they bound much less well to a Nef variant containing an engineered F9 0R mutation that interferes with docking of the native Hck RT-loop. In addi tion, we could also select RRT-SH3 domains with an opposite specificity, wh ich were dependent on the Arg90 residue for strong binding, and bound 100-f old less well to unmodified Nef. These results demonstrate the utility of p hage-display in engineering of signaling protein interaction domains, and e mphasize the importance of the RT-loop in SH3 ligand selection, thus sugges ting a general strategy for creating SH3 domains with desired binding prope rties. (C) 1999 Academic Press.