Rapid Rational Design of Cyclic Peptides Mimicking Protein-Protein Interfaces.

The cPEPmatch approach is a rapid computational methodology for the rational design of cyclic peptides to target desired regions of protein-protein interfaces. The method selects cyclic peptides that structurally match backbone structures of short segments at a protein-protein interface. In a second step, the cyclic peptides act as templates for designed binders by adapting the amino acid side chains to the side chains found in the target complex. A link to access the different tools that comprise the cPEPmatch method and a detailed step-by-step guide is provided. We outline the protocol by following the application to a trypsin protease in complex with the bovine inhibitor protein (BPTI). An extension of our original approach is also presented, where we give a detailed description of the usage of the cPEPmatch methodology focusing on identifying hot regions of protein-protein interfaces prior to the matching. This extension allows one to reduce the amount of evaluated putative cyclic peptides and to specifically design only those that compete with the strongest protein-protein binding regions. It is illustrated by an application to an MHC class I protein complex.

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster.

Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.

Rapid in silico Design of Potential Cyclic Peptide Binders Targeting Protein-Protein Interfaces.

Rational design of specific inhibitors of protein-protein interactions is desirable for drug design to control cellular signal transduction but also for studying protein-protein interaction networks. We have developed a rapid computational approach to rationally design cyclic peptides that potentially bind at desired regions of the interface of protein-protein complexes. The methodology is based on comparing the protein backbone structure of short peptide segments (epitopes) at the protein-protein interface with a collection of cyclic peptide backbone structures. A cyclic peptide that matches the backbone structure of the segment is used as a template for a binder by adapting the amino acid side chains to the side chains found in the target complex. For a small library of cyclic peptides with known high resolution structures we found for the majority (~82%) of 154 protein-protein complexes at least one very well fitting match for a cyclic peptide template to a protein-protein interface segment. The majority of the constructed protein-cyclic peptide complexes was very stable during Molecular Dynamics simulations and showed an interaction energy score that was typically more favorable compared to interaction scores of typical peptide-protein complexes. Our cPEPmatch approach could be a promising approach for rapid suggestion of cyclic peptide binders that could be tested experimentally and further improved by chemical modification.