Accumulating evidence shows that many particular proteins have evolved to bind multiple targets, including other proteins, peptides, DNA, and small molecule substrates. Multispecific recognition might be not only common but also necessary for the robustness of signaling and metabolic networks in the cell. It is also important for the immune response and for regulation of transcription and translation. Multispecificity presents an apparent paradox: How can a protein encoded by a single sequence accommodate numerous targets? Analysis of sequences and structures of multispecific proteins revealed a number of mechanisms that achieve multispecificity. Interestingly, similar mechanisms appear in antibody-antigen, T-cell receptor-peptide, protein-DNA, enzyme-substrate, and protein-protein complexes. Directed evolution and protein design experiments with multispecific proteins offer some interesting insights into the evolution of such proteins and help in the dissection of molecular interactions that mediate multispecificity. Understanding the basic principles governing multispecificity could greatly assist in the unraveling of various complex processes in the cell. In addition, through manipulation of functional multispecificity, novel proteins could be created for use in various biotechnological and biomedical applications.
Our understanding of protein evolution would greatly benefit from mapping of binding landscapes, i.e., changes in protein-protein binding affinity due to all single mutations. However, experimental generation of such landscapes is a tedious task due to a large number of possible mutations. Here, we use a simple computational protocol to map the binding landscape for two homologous high-affinity complexes, involving a snake toxin fasciculin and acetylcholinesterase from two different species. To verify our computational predictions, we experimentally measure binding between 25 Fas mutants and the 2 enzymes. Both computational and experimental results demonstrate that the Fas sequence is close to the optimum when interacting with its targets, yet a few mutations could further improve Kd, kon, and koff. Our computational predictions agree well with experimental results and generate distributions similar to those observed in other high-affinity PPIs, demonstrating the potential of simple computational protocols in capturing realistic binding landscapes.
Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein-ligand interactions. Such mimics of protein binding sites can currently be generated through structure-based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin-2 (Fas) because the structure and function of this complex is well understood. Structure-based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE-specific peptide-Fas interaction.
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