Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface

Abstract

The interactions between pro- and anti-apoptotic members of the Bcl-2 class of proteins control whether a cell lives or dies, and the study of these protein–protein interactions has been an area of intense research. In this report, we describe a new tool for the study and engineering of apoptotic protein interactions that is based on the flow cytometric detection of these interactions on the surface of Escherichia coli. After validation of the assay with the well-studied interaction between the Bak(72–87) peptide and the anti-apoptotic protein Bcl-xL, the effect of both increasing and decreasing Bak peptide length on Bcl-xL binding was investigated. Previous work demonstrated that the Bak(72–87) peptide also binds to the anti-apoptotic protein Bcl-2, albeit with lower binding affinity compared to Bcl-xL. Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72–89 of Bak binds Bcl-xL and Bcl-2 equally well. Approximate binding affinity calculations on these peptide–protein complexes confirm the experimental observations. The flow cytometric assay was also used to screen a saturation mutagenesis library of Bak(72–87) variants for improved affinity to Bcl-xL. The best variants obtained from this library exhibit an apparent Kd to Bcl-xL 4-fold lower than that of wild-type Bak(72–87).

Bcl-2 family interactome analysis using bacterial surface display

“…[42][43][44][45] Our group has developed a new technique for the analysis of these interactions based on E. coli cell surface display of a BH3 peptide and flow cytometric detection of interactions of the peptide with its anti-apoptotic binding partners. 46 Shown schematically in Fig. 1, our approach involves anchoring of the BH3 peptide to the outer membrane of E. coli via fusion of the peptide to an engineered outer membrane protein, enhanced circularly permuted OmpX (eCPX).…”

“…We have previously used this approach to investigate the binding of different lengths of Bak BH3 peptides to Bcl-x L and Bcl-2 as well as to screen a library of Bak peptide variants for improved affinity to Bcl-x L . 46 Keating and colleagues have subsequently published a similar approach using yeast cell surface display. 49 These approaches involve fusion of the BH3 peptide to another protein, thus constraining either the N-or the C-terminus of the peptide.…”

“…Despite this constraint on the peptide, our approach is able to determine apparent K d values in good agreement with K d values determined by other means. 46 Here we have utilized our E. coli surface display approach to assemble a quantitative data set comprising the interactome of nearly all human BH3 peptides with each of the five anti-apoptotic proteins. Based on these data, we have also performed extensive truncation analysis of the Bim BH3 peptide, which binds with high affinity to each of the five anti-apoptotic proteins.…”

Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein

“…The first step involved a rank‐ordered list of sequences which can fold into a given template structure prior to validation step which involved calculating fold specificity and/or binding affinity . Protein WISDOM has been employed to design peptide inhibitors for HIV‐1 entry to CD4 and Bak inhibitors of Bcl‐x L and Bcl‐2 . Meanwhile, the RosettaDesign server searched for sequences which can pack well and satisfy the hydrogen bonding potential of polar atoms for any given target structure or complex .…”

Biodiscovery of aluminum binding peptides

“…binders for various protein targets [29][30][31][32][33][34][35][36][37][38][39][40] , including some through semi-automated methods. To our knowledge, the eCPX peptide display system has never before been used in biopanning to develop affinity peptides for any bulk solid.…”