Minimizing a binding domain from protein A

We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle 159 residues total) that binds tightly (K-d = 10 nM) to the F-c portion of an immunoglobin IgG(1). Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (K-d > 1 mM), By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG(1) with a K-d Of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with F-c (the interface), As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold, Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones, Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics, Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.