Design of protein function leaps by directed domain interface evolution

Abstract

Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting ''affinity clamp'' had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution.affinity reagent ͉ epitope ͉ molecular evolution ͉ protein design ͉ PDZ domain D irected evolution-based protein engineering creates new protein functions by exploiting processes that occur during natural evolution of proteins. Protein evolution progresses via point mutations, duplication, and recombination of genes under selective pressure. The processes of gene duplication and subsequent sequence divergence (1) have been successfully recapitulated in directed evolution and computational protein design (2, 3) where preexisting active sites within natural protein scaffolds are altered to produce new functions (Fig.

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

“…Here, protein design may aim at the creation of novel domain interfaces possibly with enzymatic function or adaptation of interfaces of oligomerization or repeating unit assembly that are perturbed when steric restraints are not properly taken care of in the design. [56][57][58][59][60][61][62] Immunoglobulin domains and Fv fragments are versatile modules of proven value in biotherapeutics engineering. 1 Our CODV design differs from all previously reported designs because we do not N-or C-terminally attach VL and VH domains to create linearly extended domain strings as in, for example, diabody (Db), single-chain variable fragment (scFv), or DVD-Ig formats.…”

Protease-based synthetic sensing and signal amplification

“…Peptide-based ligands RGSIDTWV (B1) and PQPSDTWV (B2) bind the affinity clamp with K d values of 0.6 and 5 nM, respectively, by inducing association of the PDZ and FN3 domains and structuring of the connecting linker (27,28). To create a peptide-responsive version of TVMV, we replaced the protease-cleavable linker in the AI-TVMV module with the ePDZb1 version of the affinity clamp (27). As shown in Fig.…”

“…To this end, we sought to incorporate a receptor scaffold that undergoes a large conformational transition on ligand binding between the TVMV's catalytic and AI domains. As a model receptor, we chose an "affinity clamp"-an artificial two-domain receptor composed of a circularly permutated Erbin PDZ domain connected by a flexible serine-glycine linker to an engineered fibronectin type III (FN3) domain (27,28). Peptide-based ligands RGSIDTWV (B1) and PQPSDTWV (B2) bind the affinity clamp with K d values of 0.6 and 5 nM, respectively, by inducing association of the PDZ and FN3 domains and structuring of the connecting linker (27,28).…”

“…As a model receptor, we chose an "affinity clamp"-an artificial two-domain receptor composed of a circularly permutated Erbin PDZ domain connected by a flexible serine-glycine linker to an engineered fibronectin type III (FN3) domain (27,28). Peptide-based ligands RGSIDTWV (B1) and PQPSDTWV (B2) bind the affinity clamp with K d values of 0.6 and 5 nM, respectively, by inducing association of the PDZ and FN3 domains and structuring of the connecting linker (27,28). To create a peptide-responsive version of TVMV, we replaced the protease-cleavable linker in the AI-TVMV module with the ePDZb1 version of the affinity clamp (27).…”

Synthetic circuits, devices and modules

“…In some cases, creation of multi-domain proteins is not just to bring together individual domains because dramatic changes may occur at their newly formed interfaces. Huang et al found that the optimization of the interface between PDZ and FN3 domains by combinatorial library selection of FN3 loops using phage display could create new binding interface that contributes to a dramatic increase of binding affinity and specificity (Huang et al, 2008).…”