High‐resolution insights into binding of unfolded polypeptides by the PPIase chaperone SlpA

Abstract

SlpA is a 2-domain protein consisting of an FK506-binding protein (FKBP) domain that harbors the peptidyl-prolyl cis/trans-isomerase (PPIase) active site and a small insert-in-flap (IF) domain that endows the protein with chaperone activity. We have determined the structure of SlpA from Escherichia coli at 1.35-Å resolution. The overall structure is similar to other known structures of the FKBP-IF subfamily. However, by serendipity, the linker region of the purification tag binds in the chaperone binding groove of the IF domain, making this the first structure of an FKBP-IF protein in complex with a mimic of an unfolded chaperone substrate. The linker binds by β-sheet augmentation, thus completing the incomplete β barrel of the IF domain and shielding a considerable hydrophobic surface area from the solvent. Interestingly, a proline residue in trans configuration appears to be specifically recognized in a small pocket within the binding groove. Hence, the IF domain can preselect and prealign substrates with proline residues, which may explain how it enhances the catalytic efficiency and modulates the specificity of the FKBP domain in addition to its chaperone function. Based on pulldown results, we suggest that SlpA is likely to be involved in ribosome assembly.

Molecular insights into substrate recognition and catalytic mechanism of the chaperone and FKBP peptidyl-prolyl isomerase SlyD

“…1a–d . We mainly used 15-residue-long proline-containing segments from proteins that have previously been shown to bind to TtSlyD and/or other proteins from the SlyD family, namely RNase T1, which is a model protein for folding studies, and the ribosomal proteins S2 and S3 [ 17 , 18 , 24 ]; see Table 1 for the complete list of peptide sequences. Table 2 summarizes the results of the binding studies.…”

“…The stoichiometries were, however, significantly higher than 1 after fitting to a single binding site model, indicating that these peptides may still engage both binding sites (Table 2 ). We also measured binding of an eight-residue-long peptide representing the plasmid-derived linker sequence bound to the IF domain in the crystal structure of SlpA (SlpA linker; [ 18 ]), which was found to bind with an affinity similar to that of the seven-residue-long S2-short8 peptide. We conclude that the sequence requirements for peptide binding to TtSlyD FL are rather lax, which is in part because the enthalpic losses incurred by apparent sequence mismatching are readily compensated by gains in entropy, and that the affinity of binding to both domains is sensitive to the length of the peptide (Table 2 ).…”

“…The low affinity of these peptides probably relates to the smaller interaction surface compared to protein substrates, but could also relate to their limited capacity to form naturally occurring structural elements, such as β-turns. Structural insights into how substrates interact with the IF domain have so far been based on a single structure of the SlyD homologue SlpA from E. coli , in which an uncleaved purification tag is bound at the substrate binding site of the IF domain [ 18 ].…”

Plant Immunophilins: A Protein Family with Diverse Functions Beyond Protein Folding Activity

“…It consists of an FKBP-type PPIase domain and a small insert-in-fl ap (IF) domain which are responsible for its chaperone activity. It is suggested that SlpA is involved in ribosome assembly process (Quistgaard et al 2012 ).…”

Lactobacillus slpA promotes ESC growth through the ERK1/2 pathway

“…Bacterial adhesion to epithelial and subepithelial tissue is an important initial event in successful colonization of the mammalian intestine and other tissue sites. Several adhesion molecules have been characterized for bacterial species that cause infectious diseases in humans or animals (Xiao et al 2013;Quistgaard et al 2012). However, knowledge is very limited regarding adhesion molecules present on the mammalian commensal genus Lactobacillus.…”