Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron.

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

A binary effector module secreted by a type VI secretion system.

Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring cells. Cargo effectors are secreted by binding noncovalently to the T6SS apparatus. Occasionally, effector secretion is assisted by an adaptor protein, although the adaptor itself is not secreted. Here, we report a new T6SS secretion mechanism, in which an effector and a co-effector are secreted together. Specifically, we identify a novel periplasm-targeting effector that is secreted together with its co-effector, which contains a MIX (marker for type sIX effector) domain previously reported only in polymorphic toxins. The effector and co-effector directly interact, and they are dependent on each other for secretion. We term this new secretion mechanism "a binary effector module," and we show that it is widely distributed in marine bacteria.

A phage mechanism for selective nicking of dUMP-containing DNA.

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

Engineering a customizable antibacterial T6SS-based platform in Vibrio natriegens.

Bacterial pathogens are a major risk to human, animal, and plant health. To counteract the spread of antibiotic resistance, alternative antibacterial strategies are urgently needed. Here, we construct a proof-of-concept customizable, modular, and inducible antibacterial toxin delivery platform. By engineering a type VI secretion system (T6SS) that is controlled by an externally induced on/off switch, we transform the safe bacterium, Vibrio natriegens, into an effective antibacterial weapon. Furthermore, we demonstrate that the delivered effector repertoire, and thus the toxicity range of this platform, can be easily manipulated and tested. We believe that this platform can serve as a foundation for novel antibacterial bio-treatments, as well as a unique tool to study antibacterial toxins.

Significance of Individual Domains of ClpL: A Novel Chaperone from Streptococcus mutans.

ClpL is a member of the HSP100 family of AAA+ chaperones that is widely present in Gram-positive but surprisingly absent in Gram-negative bacteria. ClpL is involved in various cellular processes, including stress tolerance response, long-term survival, virulence, and antibiotic resistance. ClpL is poorly characterized, and its molecular mechanisms of chaperone activity are largely unclear. Here, we biochemically characterized the ClpL protein from Streptococcus mutans, a dental pathogen, to understand its biological functions. ClpL harbors five domains: N-domain, two nucleotide binding domains (NBD-1 and NBD-2), M-domain, and C-domain. NBD-1 and NBD-2 contain distinct Walker A and B motifs for ATP binding and hydrolysis, respectively. We found that ClpL predominantly exists as a trimer in solution; however, upon ATP binding, it rapidly forms a hexameric structure. To study structure-function activity, we constructed several substitution and deletion mutants. We found that mutations in the Walker A and B motifs interfered with ATP hydrolysis and oligomerization. Similarly, deletions of N-, M-, and C-domains abolished both ATPase activity and oligomerization. Because we previously found that ClpL acts as a chaperone, we analyzed the chaperone activity. Surprisingly, we found that the NBD-2 mutants did not display any chaperone activity, indicating that ATP binding and hydrolysis by NBD-2 are essential for the chaperone. However, NBD-1 mutants showed chaperone activities, but the activities were variable depending on the nature of the mutations. Our results indicate that unlike other HSP100 family chaperones, ClpL is a novel chaperone that does not require any additional secondary chaperones for its activity.

Dual Membrane-spanning Anti-Sigma Factors Regulate Vesiculation in Gut Bacteroidota.

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are at least partially mediated by O uter M embrane V esicles (OMVs). In this work, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron ( Bt ). Our screening led us to the identification of a novel family of D ual M embrane-spanning A nti-sigma factors (Dma), which regulate OMV biogenesis in Bt . We employed molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, results in hypervesiculation by modulating the expression of NigD1, which belongs to a family of uncharacterized proteins found throughout Bacteroidota. Dma1 has an unprecedented domain organization: it contains a C-terminal ß-barrel embedded in the OM; its N-terminal domain interacts with its cognate sigma factor in the cytoplasm, and both domains are tethered via an intrinsically disordered region that traverses the periplasm. Phylogenetic analyses reveal that the Dma family is a unique feature of Bacteroidota. This study provides the first mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

The helix located between the two domains of a mip-like peptidyl-prolyl cis-trans isomerase is crucial for its structure, stability, and protein folding ability.

FKBP22, a PPIase (peptidyl-prolyl cis-trans isomerase) produced by Escherichia coli, binds FK506 and rapamycin (both immunosuppressive drugs), shares significant homology with the Mip-like virulence factors, and has been thought to carry a long α-helix (namely α3) between its two domains. To understand whether the length of helix α3 plays any role in the structure, function, and stability of FKBP22-like proteins, we studied a recombinant E. coli FKBP22 (rFKBP22) and its four helix α3 mutant variants by various in vitro probes. Of the helix α3 mutants, two were deletion mutants (rFKBP22D5 and rFKBP22D30), whereas the two others were insertion mutants (rFKBP22I3 and rFKBP22I6). Our investigations revealed that the molecular dimensions, dimerization efficiencies, secondary structures, tertiary structures, stabilities, and protein folding abilities of all mutant proteins are different from those of rFKBP22. Conversely, the rapamycin binding affinities of the mutant proteins were affected very little. Urea-induced unfolding of each protein followed a two-state mechanism and was reversible in nature. Interestingly, rFKBP22D30 was the least stable, whereas rFKBP22I3 appeared to be the most stable of the five proteins. The data together suggest that length of helix α3 contributes significantly to the preservation of the structure, function, and stability of E. coli FKBP22.

Domain structure and denaturation of a dimeric Mip-like peptidyl-prolyl cis-trans isomerase from Escherichia coli.

FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).

Multiple T6SSs, Mobile Auxiliary Modules, and Effectors Revealed in a Systematic Analysis of the Vibrio parahaemolyticus Pan-Genome.

Type VI secretion systems (T6SSs) play a major role in interbacterial competition and in bacterial interactions with eukaryotic cells. The distribution of T6SSs and the effectors they secrete vary between strains of the same bacterial species. Therefore, a pan-genome investigation is required to better understand the T6SS potential of a bacterial species of interest. Here, we performed a comprehensive, systematic analysis of T6SS gene clusters and auxiliary modules found in the pan-genome of Vibrio parahaemolyticus, an emerging pathogen widespread in marine environments. We identified 4 different T6SS gene clusters within genomes of this species; two systems appear to be ancient and widespread, whereas the other 2 systems are rare and appear to have been more recently acquired via horizontal gene transfer. In addition, we identified diverse T6SS auxiliary modules containing putative effectors with either known or predicted toxin domains. Many auxiliary modules are possibly horizontally shared between V. parahaemolyticus genomes, since they are flanked by DNA mobility genes. We further investigated a DUF4225-containing protein encoded on an Hcp auxiliary module, and we showed that it is an antibacterial T6SS effector that exerts its toxicity in the bacterial periplasm, leading to cell lysis. Computational analyses of DUF4225 revealed a widespread toxin domain associated with various toxin delivery systems. Taken together, our findings reveal a diverse repertoire of T6SSs and auxiliary modules in the V. parahaemolyticus pan-genome, as well as novel T6SS effectors and toxin domains that can play a major role in the interactions of this species with other cells. IMPORTANCE Gram-negative bacteria employ toxin delivery systems to mediate their interactions with neighboring cells. Vibrio parahaemolyticus, an emerging pathogen of humans and marine animals, was shown to deploy antibacterial toxins into competing bacteria via the type VI secretion system (T6SS). Here, we analyzed 1,727 V. parahaemolyticus genomes and revealed the pan-genome T6SS repertoire of this species, including the T6SS gene clusters, horizontally shared auxiliary modules, and toxins. We also identified a role for a previously uncharacterized domain, DUF4225, as a widespread antibacterial toxin associated with diverse toxin delivery systems.

A DNase Type VI Secretion System Effector Requires Its MIX Domain for Secretion.

Gram-negative bacteria often employ the type VI secretion system (T6SS) to deliver diverse cocktails of antibacterial effectors into rival bacteria. In many cases, even when the identity of the delivered effectors is known, their toxic activity and mechanism of secretion are not. Here, we investigate VPA1263, a Vibrio parahaemolyticus T6SS effector that belongs to a widespread class of polymorphic effectors containing a MIX domain. We reveal a C-terminal DNase toxin domain belonging to the HNH nuclease superfamily, and we show that it mediates the antibacterial toxicity of this effector during bacterial competition. Furthermore, we demonstrate that the VPA1263 MIX domain is necessary for T6SS-mediated secretion and intoxication of recipient bacteria. These results are the first indication of a functional role for MIX domains in T6SS secretion. IMPORTANCE Specialized protein delivery systems are used during bacterial competition to deploy cocktails of toxins that target conserved cellular components. Although numerous toxins have been revealed, the activity of many remains unknown. In this study, we investigated such a toxin from the pathogen Vibrio parahaemolyticus. Our findings indicate that the toxin employs a DNase domain to intoxicate competitors. We also show that a domain used as a marker for secreted toxins is required for secretion of the toxin via a type VI secretion system.

Regions and residues of an asymmetric operator DNA interacting with the monomeric repressor of temperate mycobacteriophage L1.

Previously, the repressor protein of mycobacteriophage L1 bound to two operator DNAs with dissimilar affinity. Surprisingly, the putative operator consensus sequence, 5'GGTGGa/cTGTCAAG, lacks the dyad symmetry reported for the repressor binding operators of lambda and related phages. To gain insight into the structure of the L1 repressor-asymmetric operator DNA complex, we have performed various in vitro experiments. A dimethyl sulfate protection assay revealed that five guanine bases, mostly distributed in the two adjacent major grooves of the 13 bp operator DNA helix, participate in repressor binding. Hydroxyl radical footprinting demonstrated that interaction between the repressor and operator DNA is asymmetric in nature and occurs primarily through one face of the DNA helix. Genetic studies not only confirmed the results of the dimethyl sulfate protection assay but also indicated that other bases in the 13 bp operator DNA are critical for repressor binding. Interestingly, repressor that weakly induced bending in the asymmetric operator DNA interacted with this operator as a monomer. The tertiary structure of the L1 repressor-operator DNA complex therefore appears to be distinct from those of the lambdoid phages even though the number of repressor molecules per operator site closely matched that of the lambda phage system.

Characterization of an unusual cold shock protein from Staphylococcus aureus.

Of the three cold shock proteins expressed by Staphylococcus aureus, CspC is induced poorly by cold but strongly by various antibiotics and toxic chemicals. Using a purified CspC, here we demonstrate that it exists as a monomer in solution, possesses primarily ß-sheets, and bears substantial structural similarity with other bacterial Csps. Aggregation of CspC was initiated rapidly at temperatures above 40 °C, whereas, the Gibbs free energy of stabilization of CspC at 0 M GdmCl was estimated to be +1.6 kcal mol(-1), indicating a less stable protein. Surprisingly, CspC showed stable binding with ssDNA carrying a stretch of more than three thymine bases and binding with such ssDNA had not only stabilized CspC against proteolytic degradation but also quenched the fluorescence intensity from its exposed Trp residue. Analysis of quenching data indicates that each CspC molecule binds with ∼5 contiguous thymine bases of the above ssDNA and binding is cooperative in nature.

A novel class of polymorphic toxins in Bacteroidetes.

Bacteroidetes are Gram-negative bacteria that are abundant in the environment as well as in the gut microbiota of animals. Many bacteroidetes encode large proteins containing an N-terminal domain of unknown function, named TANFOR. In this work, we show that TANFOR-containing proteins carry polymorphic C-terminal toxin domains with predicted antibacterial and anti-eukaryotic activities. We also show that a C-terminal domain that is prevalent in TANFOR-containing proteins represents a novel family of antibacterial DNase toxins, which we named BaCT (Bacteroidetes C-terminal Toxin). Finally, we discover that TANFOR-encoding gene neighborhoods are enriched with genes that encode substrates of the type IX secretion system (T9SS), which is involved in exporting proteins from the periplasm across the outer membrane. Based on these findings, we conclude that TANFOR-containing proteins are a new class of polymorphic toxins, and we hypothesize that they are T9SS substrates.

Type VI secretion system: a modular toolkit for bacterial dominance.

Bacteria use toxin delivery systems, such as the type VI secretion system (T6SS), to antagonize competitors. The T6SS transports toxins, called effectors, directly into recipient cells. In the absence of cognate immunity proteins that protect against kin-intoxication, these effectors target conserved and essential cell components resulting in growth arrest or cell death. Here, we focus on antibacterial T6SS effectors and explore their different activities, modes of delivery, and the domains and proteins that are associated with them to provide a modular and dynamic toxin arsenal. We conclude that these natural machines present a lucrative pool and platform for future antibacterial treatments.

A modular effector with a DNase domain and a marker for T6SS substrates.

Bacteria deliver toxic effectors via type VI secretion systems (T6SSs) to dominate competitors, but the identity and function of many effectors remain unknown. Here we identify a Vibrio antibacterial T6SS effector that contains a previously undescribed, widespread DNase toxin domain that we call PoNe (Polymorphic Nuclease effector). PoNe belongs to a diverse superfamily of PD-(D/E)xK phosphodiesterases, and is associated with several toxin delivery systems including type V, type VI, and type VII. PoNe toxicity is antagonized by cognate immunity proteins (PoNi) containing DUF1911 and DUF1910 domains. In addition to PoNe, the effector contains a domain of unknown function (FIX domain) that is also found N-terminal to known toxin domains and is genetically and functionally linked to T6SS. FIX sequences can be used to identify T6SS effector candidates with potentially novel toxin domains. Our findings underline the modular nature of bacterial effectors harboring delivery or marker domains, specific to a secretion system, fused to interchangeable toxins.

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.

Strain-Dependent Recognition of a Unique Degradation Motif by ClpXP in Streptococcus mutans.

Streptococcus mutans, a dental pathogen, has a remarkable ability to cope with environmental stresses. Under stress conditions, cytoplasmic proteases play a major role in controlling the stability of regulatory proteins and preventing accumulation of damaged and misfolded proteins. ClpXP, a well-conserved cytoplasmic proteolytic system, is crucial in maintaining cellular homeostasis in bacteria. ClpX is primarily responsible for recognition of substrates and subsequent translocation of unfolded substrates into the ClpP proteolytic compartment for degradation. In Escherichia coli, ClpX recognizes distinct motifs present at the C-terminal end of target proteins. However, recognition sequences for ClpXP in other bacteria, including S. mutans, are not known. In this study, using two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) analysis, we have identified several putative substrates for S. mutans ClpXP. SsbA, which encodes a small DNA binding protein, is one such substrate that is degraded by ClpXP. By sequential deletions, we found that the last 3 C-terminal amino acids, LPF, are sufficient for ClpXP-mediated degradation. Addition of LPF at the C-terminal end of green fluorescent protein (GFP) rendered the protein completely degradable by ClpXP. Alterations of this tripeptide motif impeded ClpXP-mediated degradation. However, recognition of LPF by ClpXP is highly specific to some S. mutans strains (UA159, UA130, and N3209) since not all S. mutans strains recognize the motif. We speculate that an adaptor protein is involved in either substrate recognition or substrate degradation by ClpXP. Nevertheless, this is the first report of a unique recognition sequence for ClpXP in streptococci. IMPORTANCE Regulated proteolysis in bacteria is an important biological process that maintains protein homeostasis. ClpXP, an intracellular proteolytic complex, is the primary protease that is responsible for protein turnover. While the substrates for ClpXP were identified in Escherichia coli, the substrates for vast majority of bacteria are currently unknown. In this study, we identified a unique substrate for ClpXP-mediated degradation in Streptococcus mutans, a dental pathogen. We also found that a small motif composed of 3 amino acids is sufficient for ClpXP-mediated degradation. Identification of this motif will clearly help us to understand the pathogenesis of this organism and other related pathogens.

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.

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.

Inhibitor-induced conformational stabilization and structural alteration of a mip-like peptidyl prolyl cis-trans isomerase and its C-terminal domain.

FKBP22, an Escherichia coli-encoded PPIase (peptidyl-prolyl cis-trans isomerase) enzyme, shares substantial identity with the Mip-like pathogenic factors, caries two domains, exists as a dimer in solution and binds some immunosuppressive drugs (such as FK506 and rapamycin) using its C-terminal domain (CTD). To understand the effects of these drugs on the structure and stability of the Mip-like proteins, rFKBP22 (a chimeric FKBP22) and CTD+ (a CTD variant) have been studied in the presence and absence of rapamycin using different probes. We demonstrated that rapamycin binding causes minor structural alterations of rFKBP22 and CTD+. Both the proteins (equilibrated with rapamycin) were unfolded via the formation of intermediates in the presence of urea. Further study revealed that thermal unfolding of both rFKBP22 and rapamycin-saturated rFKBP22 occurred by a three-state mechanism with the synthesis of intermediates. Intermediate from the rapamycin-equilibrated rFKBP22 was formed at a comparatively higher temperature. All intermediates carried substantial extents of secondary and tertiary structures. Intermediate resulted from the thermal unfolding of rFKBP22 existed as the dimers in solution, carried an increased extent of hydrophobic surface and possessed relatively higher rapamycin binding activity. Despite the formation of intermediates, both the thermal and urea-induced unfolding reactions were reversible in nature. Unfolding studies also indicated the considerable stabilization of both proteins by rapamycin binding. The data suggest that rFKBP22 or CTD+ could be exploited to screen the rapamycin-like inhibitors in the future.