Modeling and monitoring the effects of three partly conserved Ile residues in the dimerization domain of a Mip-like virulence factor from Escherichia coli.

FKBP22, an Escherichia coli-made peptidyl-prolyl cis-trans isomerase, has shown considerable homology with Mip-like virulence factors. While the C-terminal domain of this enzyme is used for executing catalytic function and binding inhibitor, the N-terminal domain is employed for its dimerization. To precisely determine the underlying factors of FKBP22 dimerization, its structural model, developed using a suitable template, was carefully inspected. The data show that the dimeric FKBP22, like dimeric Mip proteins, has a V-like shape. Further, it dimerizes using 40 amino acid residues including Ile 9, Ile 17, Ile 42, and Ile 65. All of the above Ile residues except Ile 9 are partly conserved in the Mip-like proteins. To confirm the roles of the partly conserved Ile residues, three FKBP22 mutants, constructed by substituting them with an Ala residue, were studied as well. The results together indicate that Ile 65 has little role in maintaining the dimeric state or enzymatic activity of FKBP22. Conversely, both Ile 17 and Ile 42 are essential for preserving the structure, enzymatic activity, and dimerization ability of FKBP22. Ile 42 in particular looks more essential to FKBP22. However, none of these two Ile residues is required for binding the cognate inhibitor. Additional computational studies also indicated the change of V-shape and the dimeric state of FKBP22 due to the Ala substitution at position 42. The ways Ile 17 and Ile 42 protect the structure, function, and dimerization of FKBP22 have been discussed at length.Communicated by Ramaswamy H. Sarma.

The cofactors and domains of a staphylococcal capsule-producing enzyme preserve its structure, stability, shape and dimerization ability.

CapF, a staphylococcal capsule-producing enzyme, binds Zn2+ ion and NADPH using its C-terminal domain (CTD) and N-terminal domain (NTD), respectively. To elucidate the roles of cofactors and domains, we have systematically investigated the related recombinant proteins, rCapF, rCTD, recombinant NTD (rNTD) and the Zn2+-free rCapF/rCTD, Apo-rCapF/Apo-rCTD. The results show that the secondary structure, tertiary structure, shape and surface hydrophobicity of Apo-rCapF and Apo-rCTD are different from those of rCapF and rCTD. The removal of Zn2+ made rCapF thermo-sensitive, whereas both rCTD and Apo-rCTD are thermo-resistant proteins. Further, Apo-rCapF and rCapF existed as the dimers, whereas rCTD and Apo-rCTD formed a mixture of dimers and tetramers in the aqueous solution. Zn2+ maintained the structure of NTD as well. The NADPH binding activity and Cys accessibility of rNTD, rCapF and Apo-rCapF were significantly different from each other. The binding of NADPH to the above three proteins freely occurred, liberated heat at 25°C and increased their diameters. In addition, the structure, stability, shape and oligomerization ability of rNTD, rCTD and rCapF little resembled each other. Collectively, the domains and cofactors of CapF contribute to preserving its conformation, stability, shape and dimerization ability.

Serine 106 preserves the tertiary structure, function, and stability of a cyclophilin from Staphylococcus aureus.

SaCyp, a staphylococcal cyclophilin involved in both protein folding and pathogenesis, has a Ser residue at position 106 and a Trp residue at position 136. While Ser 106 of SaCyp aligned with a cyclosporin A (CsA) binding Ala residue, its Trp 136 aligned with a Trp or a Phe residue of most other cyclophilins. To demonstrate the exact roles of Ser 106 and Trp 136 in SaCyp, we have elaborately studied rCyp[S106A] and rCyp[W136A], two-point mutants of a recombinant SaCyp (rCyp) harboring an Ala substitution at positions 106 and 136, respectively. Of the mutants, rCyp[W136A] showed the rCyp-like CsA binding affinity and peptidyl-prolyl cis-trans isomerase (PPIase) activity. Conversely, the PPIase activity, CsA binding affinity, stability, tertiary structure, surface hydrophobicity, and Trp accessibility of rCyp[S106A] notably differed from those of rCyp. The computational experiments also reveal that the structure, dimension, and fluctuation of SaCyp are not identical to those of SaCyp[S106A]. Furthermore, Ser at position 106 of SaCyp, compared to Ala at the same position, formed a higher number of non-covalent bonds with CsA. Collectively, Ser 106 is an indispensable residue for SaCyp that keeps its tertiary structure, function, and stability intact.Communicated by Ramaswamy H. Sarma.

A conserved arginine residue in a staphylococcal anti-sigma factor is required to preserve its kinase activity, structure, and stability.

RsbW, σB, and RsbV, encoded by Staphylococcus aureus and related bacteria, act as an anti-sigma factor, an sigma factor, and an anti-anti-sigma factor, respectively. The interaction between RsbW and σB blocks the transcription initiation activity of the latter protein. RsbW also functions as a serine kinase and phosphorylates RsbV in the presence of ATP. Our modeling study indicates that the RsbW-RsbV complex is stabilized by twenty-four intermolecular non-covalent bonds. Of the bond-forming RsbW residues, Arg 23, and Glu 49 are conserved residues. To understand the roles of Arg 23 in RsbW, rRsbW[R23A], a recombinant S. aureus RsbW (rRsbW) harboring Arg to Ala change at position 23, was investigated using various probes. The results reveal that rRsbW[R23A], like rRsbW, exists as the dimers in the aqueous solution. However, rRsbW[R23A], unlike rRsbW, neither interacted with a chimeric RsbV (rRsbV) nor formed the phosphorylated rRsbV in the presence of ATP. Furthermore, the tertiary structure and hydrophobic surface area of rRsbW[R23A] matched little with those of rRsbW. Conversely, both rRsbW[R23A] and rRsbW showed interaction with a recombinant σB (rσB). rRsbW and rRsbW[R23A] were also unfolded via the formation of at least one intermediate in the presence of urea. However, the thermodynamic stability of rRsbW significantly differed from that of rRsbW[R23A]. Our molecular dynamics (MD) simulation study also reveals the substantial change of structure, dimension, and stability of RsbW due to the above mutation. The ways side chain of critical Arg 23 contributes to maintaining the tertiary structure, and stability of RsbW was elaborately discussed.Communicated by Ramaswamy H. Sarma.

Structurally distinct unfolding intermediates formed from a staphylococcal capsule-producing enzyme retained NADPH binding activity.

CapF, a capsule-producing enzyme expressed by Staphylococcus aureus, binds NADPH and exists as a dimer in the aqueous solution. Many other capsule-producing virulent bacteria also express CapF orthologs. To understand the folding-unfolding mechanism of S. aureus CapF, herein a recombinant CapF (rCapF) was individually investigated using urea and guanidine hydrochloride (GdnCl). Unfolding of rCapF by both the denaturants was reversible but proceeded via the synthesis of a different number of intermediates. While two dimeric intermediates (rCapF4 and rCapF5) were formed at 0.5 M and 1.5 M GdnCl, three dimeric intermediates (rCapF1, rCapF2, and rCapF3) were produced at 1 M, 2 M, and 3 M urea, respectively. rCapF5 showed 3.6 fold less NADPH binding activity, whereas other intermediates retained full NADPH binding activity. Compared to rCapF, all of the intermediates (except rCapF3) had a compressed shape. Conversely, rCapF3 possessed a native protein-like shape. The maximum shape loss was in rCapF4 though its secondary structure remained unperturbed. Additionally, the tertiary structure and hydrophobic surface area of the intermediates neither matched with each other nor with those of the native rCapF. Of the four Trp residues in rCapF, one or more Trp residues in the intermediates may have higher solvent accessibility. Using sequence alignment and a tertiary structural model of CapF, we have demonstrated that the region around Trp 137 of CapF may be most sensitive to unfolding, whereas the NADPH binding motif carrying region at the N-terminal end of this protein may be resistant to unfolding, particularly at the low denaturant concentrations.Communicated by Ramaswamy H. Sarma.

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

σB, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

Understanding the structure, stability, and anti-sigma factor-binding thermodynamics of an anti-anti-sigma factor from Staphylococcus aureus.

Staphylococcus aureus and many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by σB, an alternative sigma factor. Our structural and thermodynamic studies of a recombinant S. aureus RsbV (rRsbV) show that the monomeric protein contains five α-helices and a mostly parallel but mixed ß-sheet composed of five ß-strands, and interacts with a chimeric S. aureus RsbW (rRsbW) in vitro. In addition, rRsbV binds rRsbW with a Kd of 0.058 µM using spectroscopy and 0.008 µM using calorimetry at 25 °C. From a gel-shift assay, the affinity of rRsbV to rRsbW was found to be higher than its affinity with a recombinant S. aureus σB (rσB). Moreover, the heat generated from the spontaneous rRsbV - rRsbW interaction changed in a compensatory manner with entropy in the 20°-35 °C range. The association between rRsbV and rRsbW yielded a negative heat capacity change, suggesting that both hydrogen bonds and hydrophobic interactions participate in the formation of the rRsbV-rRsbW complex. Computational analyses of a homology-based RsbV-RsbW model has mostly supported the formation of a 2: 2 complex verified by gel filtration chromatography, the experimental ΔG and the existence of these non-covalent bonds. Our unfolding experiments show that the thermodynamic stability of rRsbV is significantly increased in the presence of rRsbW. Thus, these studies have provided valuable insights into the structure, stability, and the anti-sigma-binding thermodynamics of an anti-anti-sigma factor.Communicated by Ramaswamy H. Sarma.

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape.

SaCyp, a cyclophilin having 197 amino acid residues, acts both as a protein-folding catalyst and a virulence factor in Staphylococcus aureus. Interestingly, a region, homologous to the SaCyp region carrying 121-148 amino acid residues, is present in many putative cyclophilins but absent in well-studied cyclophilins. To determine the exact roles of this unusual region in SaCyp and related proteins, we have investigated a deletion mutant (rCypΔ) of a recombinant SaCyp (rCyp) using various probes. The data reveal that rCypΔ has significantly less catalytic activity and possesses altered structure and hydrophobic surface compared to rCyp. Conversely, the deletion substantially increased inhibitor binding affinity and altered the shape of rCyp. However, both proteins were unfolded by a non-two-state mechanism in the presence of urea. Additionally, the stability of rCyp was significantly reduced due to the deletion of the residues 121-148. Our MD simulation study also indicated the considerable alteration in structure, shape, and fluctuations of SaCyp due to the removal of the region carrying 121-148 residues. Hence, the atypical region located in SaCyp might be vital for maintaining its unique structure, function, stability, and shape.

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity.

Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. Both the secondary structure and the tertiary structure of each intermediate appears very similar to those of the corresponding native protein. Conversely, the hydrophobic surface areas of the intermediates are comparatively less. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in the future.

Dimerization ability, denaturation mechanism, and the stability of a staphylococcal phage repressor and its two domains.

The lysogenic growth of phage ф11 in Staphylococcus aureus is controlled by a repressor (CI) that harbors an N-terminal domain (NTD), and a C-terminal domain (CTD). Previously, NTD, like CI, showed DNA binding activity and dimerized in the aqueous solution. To precisely understand the folding mechanism, function, and the stability of CI, NTD, and CTD, we have investigated their recombinant forms, rCI, rNTD, and rCTD, using various probes. The data reveal that rCTD, like rCI and rNTD, is a well-structured protein and produces dimers in the aqueous environment. However, the stability order of the dimers appears to be rCI > rCTD > rNTD. Interestingly, the stability of rNTD or rCTD looks slightly higher than that of rCI. The urea-induced equilibrium unfolding of these proteins proceeded via the production of two intermediates. The structure, surface hydrophobicity, and the dimeric status of one intermediate mostly differed from those of another intermediate or the native protein. Our MD simulation study on the representative NTD shows the substantial change in its structure and stability at the urea concentrations, which formed rNTD intermediates. Collectively, the computational data have supported the experimental data and indicated that the CI and its domains are folded by a similar multiphasic pathway.

NRF2 transcriptionally activates the heat shock factor 1 promoter under oxidative stress and affects survival and migration potential of MCF7 cells.

Functional up-regulation of heat shock factor 1 (HSF1) activity through different posttranslational modifications has been implicated in the survival and proliferation of various cancers. It is increasingly recognized that the HSF1 gene is also up-regulated at the transcriptional level, a phenomenon correlated with poor prognosis for patients with different cancers, including breast cancer. Here, we analyzed the transcriptional up-regulation of HSF1 in human cells upon arsenite- or peroxide-induced oxidative stress. Sequential promoter truncation coupled with bioinformatics analysis revealed that this activation is mediated by two antioxidant response elements (AREs) located between 1707 and 1530 bp upstream of the transcription start site of the HSF1 gene. Using shRNA-mediated down-regulation, ChIP of NRF2, site-directed mutagenesis of the AREs, and DNase I footprinting of the HSF1 promoter, we confirmed that nuclear factor erythroid-derived 2-like 2 (NRF2, also known as NFE2L2) interacts with these AREs and up-regulates HSF1 expression. We also found that BRM/SWI2-related gene 1 (BRG1), a catalytic subunit of SWI2/SNF2-like chromatin remodeler, is involved in this process. We further show that NRF2-dependent HSF1 gene regulation plays a crucial role in cancer cell biology, as interference with NRF2-mediated HSF1 activation compromised survival, migration potential, and the epithelial-to-mesenchymal transition and autophagy in MCF7 breast cancer cells exposed to oxidative stress. Taken together, our findings unravel the mechanistic basis of HSF1 gene regulation in cancer cells and provide molecular evidence supporting a direct interaction between HSF1 and NRF2, critical regulators of two cytoprotective mechanisms exploited by cancer cells.

A staphylococcal anti-sigma factor possesses a single-domain, carries different denaturant-sensitive regions and unfolds via two intermediates.

RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates. The structure, shape, and the surface hydrophobicity of no intermediate completely matches with those of other intermediates or the native rRsbW. Interestingly, one of the intermediates, formed in the presence of less GdnCl concentrations, has a molten globule-like structure. Conversely, all of the intermediates, like native rRsbW, exist as dimers in aqueous solution. The putative molten globule and the urea-generated intermediates also have retained some kinase activity. Additionally, the putative ATP binding site/catalytic site of rRsbW shows higher denaturant sensitivity than the tentative dimerization region of this enzyme.

Alanine substitution mutations in the DNA binding region of a global staphylococcal virulence regulator affect its structure, function, and stability.

SarA, a winged-helix DNA binding protein, is a global virulence regulator in Staphylococcus aureus. The putative DNA binding region of SarA is located between amino acid residues Leu 53 and Gln 97. Previous studies have demonstrated that residues at positions 84, 88, 89, and 90 are critical for its function. To precisely understand the roles of the DNA binding residues, we have investigated nine mutants of a recombinant SarA (rSarA) along with the rSarA mutants carrying mutations at the above four positions. Of the thirteen mutants, eleven mutants show weaker DNA binding activity in vitro compared to rSarA. As noted earlier, the DNA binding affinity of rSarA was maximally affected due to the mutation at position 84 or 90. Each of the functionally-defective mutants also possesses an altered structure and stability. Additionally, the mutations at positions 84 and 90 have severely affected the formation of hydrogen (H) bonds at the interface between SarA and the cognate DNA. The mutation at position 64 also has perturbed the generation of some interface H-bonds. Therefore, the disruption of H-bonds in the protein-DNA interface and the structural alteration in the protein may be responsible for the reduced DNA binding activity of the mutants.

Determining the Roles of a Conserved α-Helix in a Global Virulence Regulator from Staphylococcus aureus.

SarA, a pleiotropic transcription regulator, is encoded by Staphylococcus aureus, a pathogenic bacterium. The expression of many virulence and non-virulence genes in S. aureus is modulated by this regulator. Structural studies have shown it to be a winged-helix DNA-binding protein carrying two monomers. Each SarA monomer is composed of five α-helices (α1-α5), three ß-strands (ß1-ß3) and multiple loops. The putative DNA binding region of SarA is constituted with α3, α4, ß2, and ß3, whereas, its dimerization seems to occur using α1, α2, and α5. Interestingly, many SarA-like proteins are dimeric and use three or more helices for their dimerization. To clearly understand the roles of helix α1 in the dimerization, we have constructed and purified a SarA mutant (Δα1) that lacks helix α1. Our in-depth studies with Δα1 indicate that the helix α1 is critical for preserving the structure, DNA binding activity and thermodynamic stability of SarA. However, the helix has little affected its dimerization ability. Possible reasons for such anomaly have been discussed at length.

Identification and characterization of a CI binding operator at a distant location in the temperate staphylococcal phage ф11.

Bacteriophage ф11 encodes repressors CI and Cro for executing its growth in Staphylococcus aureus, a human pathogen. There are three homologous operators O1, O2 and O3 between the repressor-expressing genes. While CI binds to O1 and O2, Cro interacts only with O3. To locate additional CI binding operators in ф11, we searched its genome using the O1/O2 sequence as a probe. The results show the presence of a putative CI binding operator (O4) at the 3΄ end of the cro. O4 differs from O2 and O1 by one base and five bases, respectively. A specific interaction was noticed between O4 and rCI, a recombinant CI. However, O4 shows no interaction with rCro, a chimeric Cro. Additionally, six guanine bases, situated in and around O4, have interacted with rCI. Interestingly, the rCI binding affinity of O4 or O1 is about 15 times higher than that of O2. A comparative study indicates that some bases and structural alteration, unique to O1 and O4, may contribute to their enhanced rCI binding affinity. Collectively, the study has not only broadened the distinct gene regulatory circuit of ф11 but also suggested that it possibly employs a complex mechanism for its development in S. aureus.

Identification and characterization of a cyclosporin binding cyclophilin from Staphylococcus aureus Newman.

Cyclophilins, a class of peptidyl-prolyl cis-trans isomerase (PPIase) enzymes, are inhibited by cyclosporin A (CsA), an immunosuppressive drug. Staphylococcus aureus Newman, a pathogenic bacterium, carries a gene for encoding a putative cyclophilin (SaCyp). SaCyp shows significant homology with other cyclophilins at the sequence level. A three-dimensional model structure of SaCyp harbors a binding site for CsA. To verify whether SaCyp possesses both the PPIase activity and the CsA binding ability, we have purified and investigated a recombinant SaCyp (rCyp) using various in vitro tools. Our RNase T1 refolding assay indicates that rCyp has a substantial extent of PPIase activity. rCyp that exists as a monomer in the aqueous solution is truly a cyclophilin as its catalytic activity specifically shows sensitivity to CsA. rCyp appears to bind CsA with a reasonably high affinity. Additional investigations reveal that binding of CsA to rCyp alters its structure and shape to some extent. Both rCyp and rCyp-CsA are unfolded via the formation of at least one intermediate in the presence of guanidine hydrochloride. Unfolding study also indicates that there is substantial extent of thermodynamic stabilization of rCyp in the presence of CsA as well. The data suggest that rCyp may be exploited to screen the new antimicrobial agents in the future.

A Surfactant-Induced Functional Modulation of a Global Virulence Regulator from Staphylococcus aureus.

Triton X-100 (TX-100), a useful non-ionic surfactant, reduced the methicillin resistance in Staphylococcus aureus significantly. Many S. aureus proteins were expressed in the presence of TX-100. SarA, one of the TX-100-induced proteins, acts as a global virulence regulator in S. aureus. To understand the effects of TX-100 on the structure, and function of SarA, a recombinant S. aureus SarA (rSarA) and its derivative (C9W) have been investigated in the presence of varying concentrations of this surfactant using various probes. Our data have revealed that both rSarA and C9W bind to the cognate DNA with nearly similar affinity in the absence of TX-100. Interestingly, their DNA binding activities have been significantly increased in the presence of pre-micellar concentration of TX-100. The increase of TX-100 concentrations to micellar or post-micellar concentration did not greatly enhance their activities further. TX-100 molecules have altered the secondary and tertiary structures of both proteins to some extents. Size of the rSarA-TX-100 complex appears to be intermediate to those of rSarA and TX-100. Additional analyses show a relatively moderate interaction between C9W and TX-100. Binding of TX-100 to C9W has, however, occurred by a cooperative pathway particularly at micellar and higher concentrations of this surfactant. Taken together, TX-100-induced structural alteration of rSarA and C9W might be responsible for their increased DNA binding activity. As TX-100 has stabilized the somewhat weaker SarA-DNA complex effectively, it could be used to study its structure in the future.

Determining the roles of a conserved tyrosine residue in a Mip-like peptidyl-prolyl cis-trans isomerase.

The FKBP22 and the related peptidyl-prolyl cis-trans isomerases dimerize using their N-terminal domains. Conversely, their C-terminal domains possess both the substrate and inhibitor binding sites. To delineate the roles of a conserved Tyr residue at their N-terminal domains, we have studied a FKBP22 mutant that carries an Ala in place of the conserved Tyr at position 15. We have demonstrated that the Tyr 15 of FKBP22 is indispensable for preserving its dimerization ability, catalytic activity, and structure. The residue, however, little contributed to its inhibitor binding ability and stability. The mode of action of Tyr 15 has been discussed at length.

Chemical and thermal unfolding of a global staphylococcal virulence regulator with a flexible C-terminal end.

SarA, a Staphylococcus aureus-specific dimeric protein, modulates the expression of numerous proteins including various virulence factors. Interestingly, S. aureus synthesizes multiple SarA paralogs seemingly for optimizing the expression of its virulence factors. To understand the domain structure/flexibility and the folding/unfolding mechanism of the SarA protein family, we have studied a recombinant SarA (designated rSarA) using various in vitro probes. Limited proteolysis of rSarA and the subsequent analysis of the resulting protein fragments suggested it to be a single-domain protein with a long, flexible C-terminal end. rSarA was unfolded by different mechanisms in the presence of different chemical and physical denaturants. While urea-induced unfolding of rSarA occurred successively via the formation of a dimeric and a monomeric intermediate, GdnCl-induced unfolding of this protein proceeded through the production of two dimeric intermediates. The surface hydrophobicity and the structures of the intermediates were not identical and also differed significantly from those of native rSarA. Of the intermediates, the GdnCl-generated intermediates not only possessed a molten globule-like structure but also exhibited resistance to dissociation during their unfolding. Compared to the native rSarA, the intermediate that was originated at lower GdnCl concentration carried a compact shape, whereas, other intermediates owned a swelled shape. The chemical-induced unfolding, unlike thermal unfolding of rSarA, was completely reversible in nature.

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

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.