Proline substitutions in a Mip-like peptidyl-prolyl cis-trans isomerase severely affect its structure, stability, shape and activity

Abstract

FKBP22, an Escherichia coli-specific peptidyl-prolyl cis-trans isomerase, shows substantial homology with the Mip-like virulence factors. Mip-like proteins are homodimeric and possess a V-shaped conformation. Their N-terminal domains form dimers, whereas their C-terminal domains bind protein/peptide substrates and distinct inhibitors such as rapamycin and FK506. Interestingly, the two domains of the Mip-like proteins are separated by a lengthy, protease-susceptible α-helix. To delineate the structural requirement of this domain-connecting region in Mip-like proteins, we have investigated a recombinant FKBP22 (rFKBP22) and its three point mutants I65P, V72P and A82P using different probes. Each mutant harbors a Pro substitution mutation at a distinct location in the hinge region. We report that the three mutants are not only different from each other but also different from rFKBP22 in structure and activity. Unlike rFKBP22, the three mutants were unfolded by a non-two state mechanism in the presence of urea. In addition, the stabilities of the mutants, particularly I65P and V72P, differed considerably from that of rFKBP22. Conversely, the rapamycin binding affinity of no mutant was different from that of rFKBP22. Of the mutants, I65P showed the highest levels of structural/functional loss and dissociated partly in solution. Our computational study indicated a severe collapse of the V-shape in I65P due to the anomalous movement of its C-terminal domains. The α-helical nature of the domain-connecting region is, therefore, critical for the Mip-like proteins.

Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli

“…Such fklB mutants and the wild type were expressed in the Δ6 ppi derivative SR21984 and proteins were purified under identical conditions. Substitutions like Y15A in the N-terminal domain should render FklB in the monomeric state [ 20 , 21 ], while substitutions in C-terminal residues W158 and F198 can affect the PPIase and the chaperone-like activity. Measurement of the PPIase activity using N -Suc-Ala-Ala- cis -Pro-Phe- p -nitroanilide peptide as a substrate revealed a severe reduction of PPIase activity in the case of FklB W158Y and a loss of PPIase activity with FklB F198A mutants ( Figure 6 C).…”

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3

“…The binding of a ligand usually stabilizes a cognate protein. ,,− The equilibrium unfolding of proteins in the presence and absence of cognate ligands has long been exploited to obtain such clues. , To see whether binding of rRsbW stabilizes rσ B3 , we have investigated the urea-induced unfolding of rRsbW equilibrated/unequilibrated FITC-rσ B3 by a fluorimeter as earlier stated . The recorded fluorescence spectra of rRsbW-bound/unbound FITC-rσ B3 show that the fluorescence intensities are gradually increased upon raising the urea concentrations from 0 to 7 M (Figure S5).…”

A comprehensive study of the voltage gated potassium channel Kv4.3: from functional analysis to molecular dynamics modelling

“…We may speculate that the substitution of L310 to proline may hinder the folding of the Kv4.3 channel subunit by breaking the α-helical domain in which L310 resides. Substitutions of amino acids to proline have already been shown to affect the structure and protein folding ability, as was reported for Mip-like peptidyl-prolyl cis-trans isomerase (rFKBP22) (Polley, Chakravarty, Chakrabarti, Chattopadhyaya, & Sau, 2015). Moreover, given that we were not able to use MD simulations or perform electrophysiology recordings on the L310P mutant channel we advocate for the need to develop a method to investigate the electrical activity of the Kv4.3 channel in a cell-free environment (see Chapter 6).…”