Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Abstract

We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/ selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.calorimetry ͉ intrabody ͉ ribosome display ͉ Sac7d ͉ type II secretion system

Type II Secretion System Secretin PulD Localizes in Clusters in theEscherichia coliOuter Membrane

“…Sac7*40 is an artificial protein (affitin) that binds specifically and with high affinity to an unidentified epitope in the N-terminal half of PulD (22). Incubation of fixed and permeabilized PAP7232 cells with Sac7*40-GFP followed by fluorescence microscopy showed the presence of zero to four bright foci per cell and a weaker fluorescence of the whole cell body.…”

“…For localization of PulD using the purified Sac7*40-GFP fusion protein (22), cells were resuspended in phosphate-buffered saline (PBS) and immobilized on polylysine-coated glass coverslips at room temperature for 30 min. After the coverslips were washed two times with PBS, the cells were fixed in 2% formaldehyde in PBS for 30 min.…”

“…In this work, we examined the cellular localization of the secretin PulD as a chimera with a stable variant of monomeric red fluorescent protein (RFP), mCherry (31), and studied the effect of its pilotin, PulS, and other secreton components on its membrane localization and organization. To validate the use of PulD-mCherry as a model for localizing PulD in the envelope, we also introduced the use of Sac7d-derived binding proteins (22), hereafter called affitins. GFP-tagged affitins can be used as alternatives for fluorescein-labeled antibodies in immunofluorescence-like tests.…”

Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability

“…Thus, the combinatorial approach, which consists of generating random variants coupled to a screening for stability, and methods based on sequence/structure homology have proved to be some of the most successful strategies to improve the stability of numerous proteins (for reviews, see (Bommarius et al, 2006;Romero and Arnold, 2009) Affitins are artificial affinity proteins that we have developed which are derived from extremophilic proteins from the "7 kDa DNA-binding" family found in Archaea, such as Sac7d (Béhar et al, 2013;Buddelmeijer et al, 2009;Krehenbrink et al, 2008;Mouratou et al, 2007). Sac7d is a hyperthermostable protein (T m = 90.4 • C) (Edmondson and Shriver, 2001;McCrary et al, 1996;Mouratou et al, 2007) and is chemically resistant from pH 0 up to pH 12 (Béhar et al, 2013). Binders with high pH and thermal stabilities have been obtained by coupling the generation of combinatorial libraries of Sac7d variants corresponding to the randomization of 10-14 residues of the ␤-sheet surface originally involved in the binding of DNA and selections against different targets.…”

Parallel Processing of Complex Biomolecular Information: Combining Experimental and Computational Approaches

“…Cycles of selection and recovery can be iterated both to enrich rare ligand-binding molecules, and to select molecules with the best affinity. Ribosome display has been used for the selection of proteins, such as scFv antibody fragments and alternative binding scaffolds with specificity and affinity to peptides (Hanes & Pluckthun, 1997), proteins (Hanes et al, 2000;Knappik et al, 2000;Binz et al, 2004;Lee et al, 2004;Mouratou et al, 2007) and nucleic acids (Schaffitzel et al, 2001). Using transitionstate analogs or enzyme inhibitors that bind reversibly to their enzyme (suicide substrates), ribosome display can also be used for the selection for enzymatic activity.…”