Co-phase separation of Y14 and RNA in vitro and its implication for DNA repair.

The multifunctional RNA recognition motif-containing protein Y14/RBM8A participates in mRNA metabolism and is essential for the efficient repair of DNA double-strand breaks (DSBs). Y14 contains highly charged, low-complexity sequences in both the amino- and carboxy-terminal domains. The feature of charge segregation suggests that Y14 may undergo liquid-liquid phase separation (LLPS). Recombinant Y14 formed phase-separated droplets, which were sensitive to pH and salt concentration. Domain mapping suggested that LLPS of Y14 involves multivalent electrostatic interactions and is partly determined by the net charge of its low-complexity regions. Phospho-mimicry of the carboxy-terminal arginine-serine dipeptides of Y14 suppressed phase separation. Moreover, RNA could phase separate into Y14 droplets and modulate Y14 LLPS in a concentration-dependent manner. Finally, the capacity of Y14 in LLPS and coacervation with RNA in vitro correlated with its activity in DSB repair. These results reveal a molecular rule for LLPS of Y14 in vitro and an implication for its co-condensation with RNA in genome stability.

Structural basis of transcriptional activation by the OmpR/PhoB-family response regulator PmrA.

PmrA, an OmpR/PhoB-family response regulator, triggers gene transcription responsible for polymyxin resistance in bacteria by recognizing promoters where the canonical-35 element is replaced by the pmra-box, representing the PmrA recognition sequence. Here, we report a cryo-electron microscopy (cryo-EM) structure of a bacterial PmrA-dependent transcription activation complex (TAC) containing a PmrA dimer, an RNA polymerase σ70 holoenzyme (RNAPH) and the pbgP promoter DNA. Our structure reveals that the RNAPH mainly contacts the PmrA C-terminal DNA-binding domain (DBD) via electrostatic interactions and reorients the DBD three base pairs upstream of the pmra-box, resulting in a dynamic TAC conformation. In vivo assays show that the substitution of the DNA-recognition residue eliminated its transcriptional activity, while variants with altered RNAPH-interacting residues resulted in enhanced transcriptional activity. Our findings suggest that both PmrA recognition-induced DNA distortion and PmrA promoter escape play crucial roles in its transcriptional activation.

Structural basis for promoter DNA recognition by the response regulator OmpR.

OmpR, a response regulator of the EnvZ/OmpR two-component system (TCS), controls the reciprocal regulation of two porin proteins, OmpF and OmpC, in bacteria. During signal transduction, OmpR (OmpR-FL) undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (OmpRn) and recognizes the promoter DNA from its C-terminal DNA-binding domain (OmpRc) to elicit an adaptive response. Apart from that, OmpR regulates many genes in Escherichia coli and is important for virulence in several pathogens. However, the molecular mechanism of the regulation and the structural basis of OmpR-DNA binding is still not fully clear. In this study, we presented the crystal structure of OmpRc in complex with the F1 region of the ompF promoter DNA from E. coli. Our structural analysis suggested that OmpRc binds to its cognate DNA as a homodimer, only in a head-to-tail orientation. Also, the OmpRc apo-form showed a unique domain-swapped crystal structure under different crystallization conditions. Biophysical experimental data, such as NMR, fluorescent polarization and thermal stability, showed that inactive OmpR-FL (unphosphorylated) could bind to promoter DNA with a weaker binding affinity as compared with active OmpR-FL (phosphorylated) or OmpRc, and also confirmed that phosphorylation may only enhance DNA binding. Furthermore, the dimerization interfaces in the OmpRc-DNA complex structure identified in this study provide an opportunity to understand the regulatory role of OmpR and explore the potential for this "druggable" target.

Structural basis for -35 element recognition by σ4 chimera proteins and their interactions with PmrA response regulator.

In class II transcription activation, the transcription factor normally binds to the promoter near the -35 position and contacts the domain 4 of σ factors (σ4 ) to activate transcription. However, σ4 of σ70 appears to be poorly folded on its own. Here, by fusing σ4 with the RNA polymerase ß-flap-tip-helix, we constructed two σ4 chimera proteins, one from σ70σ470c and another from σSσ4Sc of Klebsiella pneumoniae. The two chimera proteins well folded into a monomeric form with strong binding affinities for -35 element DNA. Determining the crystal structure of σ4Sc in complex with -35 element DNA revealed that σ4Sc adopts a similar structure as σ4 in the Escherichia coli RNA polymerase σS holoenzyme and recognizes -35 element DNA specifically by several conserved residues from the helix-turn-helix motif. By using nuclear magnetic resonance (NMR), σ470c was demonstrated to recognize -35 element DNA similar to σ4Sc . Carr-Purcell-Meiboom-Gill relaxation dispersion analyses showed that the N-terminal helix and the ß-flap-tip-helix of σ470c have a concurrent transient α-helical structure and DNA binding reduced the slow dynamics on σ470c . Finally, only σ470c was shown to interact with the response regulator PmrA and its promoter DNA. The chimera proteins are capable of -35 element DNA recognition and can be used for study with transcription factors or other factors that interact with domain 4 of σ factors.

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis.

Solution structure and tandem DNA recognition of the C-terminal effector domain of PmrA from Klebsiella pneumoniae.

Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrAC and characterized the interactions between PmrAC or BeF3(-)-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC binds to the PmrA box, which was verified to contain two half-sites, 5'-CTTAAT-3' and 5'-CCTAAG-3', in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrAC-DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF-DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation.

Structural basis of a physical blockage mechanism for the interaction of response regulator PmrA with connector protein PmrD from Klebsiella pneumoniae.

In bacteria, the two-component system is the most prevalent for sensing and transducing environmental signals into the cell. The PmrA-PmrB two-component system, responsible for sensing external stimuli of high Fe(3+) and mild acidic conditions, can control the genes involved in lipopolysaccharide modification and polymyxin resistance in pathogens. In Klebsiella pneumoniae, the small basic connector protein PmrD protects phospho-PmrA and prolongs the expression of PmrA-activated genes. We previously determined the phospho-PmrA recognition mode of PmrD. However, how PmrA interacts with PmrD and prevents its dephosphorylation remains unknown. To address this question, we solved the x-ray crystal structure of the N-terminal receiver domain of BeF3(-)-activated PmrA (PmrA(N)) at 1.70 Å. With this structure, we applied the data-driven docking method based on NMR chemical shift perturbation to generate the complex model of PmrD-PmrA(N), which was further validated by site-directed spin labeling experiments. In the complex model, PmrD may act as a blockade to prevent phosphatase from contacting with the phosphorylation site on PmrA.

Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal ß-hairpin conformation.

Trichomonas vaginalis Myb3 transcription factor (tvMyb3) recognizes the MRE-1 promoter sequence and regulates ap65-1 gene, which encodes a hydrogenosomal malic enzyme that may play a role in the cytoadherence of the parasite. Here, we identified tvMyb3(53-180) as the essential fragment for DNA recognition and report the crystal structure of tvMyb3(53-180) bound to MRE-1 DNA. The N-terminal fragment adopts the classical conformation of an Myb DNA-binding domain, with the third helices of R2 and R3 motifs intercalating in the major groove of DNA. The C-terminal extension forms a ß-hairpin followed by a flexible tail, which is stabilized by several interactions with the R3 motif and is not observed in other Myb proteins. Interestingly, this unique C-terminal fragment does not stably connect with DNA in the complex structure but is involved in DNA binding, as demonstrated by NMR chemical shift perturbation, (1)H-(15)N heteronuclear-nuclear Overhauser effect and intermolecular paramagnetic relaxation enhancement. Site-directed mutagenesis also revealed that this C-terminal fragment is crucial for DNA binding, especially the residue Arg(153) and the fragment K(170)KRK(173). We provide a structural basis for MRE-1 DNA recognition and suggest a possible post-translational regulation of tvMyb3 protein.

Structural insights into the role of N-terminal integrity in PhoSL for core-fucosylated N-glycan recognition.

PhoSL (Pholiota squarrosa Lectin) has an exceptional binding affinity for biomolecules with core-fucosylated N-glycans. This modification involves the addition of fucose to the inner N-acetylglucosamine within the N-glycan structure and is known to influence many physiological processes. Nevertheless, the molecular interactions underlying high-affinity binding of native PhoSL to core-fucosylated N-glycans remain largely unknown. In this study, we devised a strategy to produce PhoSL with the essential structural characteristics of the native protein (n-PhoSL). To do so, a fusion protein was expressed in E. coli and purified. Then, enzymatic cleavage and incubation with glutathione were utilized to recapitulate the native primary structure and disulfide bonding pattern. Subsequently, we identified the residues crucial for n-PhoSL binding to core-fucosylated chitobiose (N2F) via NMR spectroscopy. Additionally, crystal structures were solved for both apo n-PhoSL and its N2F complex. These analyses suggested a pivotal role of the N-terminal amine in maintaining the integrity of the binding pocket and actively contributing to core-fucose recognition. In support of this idea, the inclusion of additional residues at the N-terminus considerably reduced binding affinity and PhoSL cytotoxicity toward breast cancer cells. Taken together, these findings can facilitate the utilization of PhoSL in basic research, diagnostics and therapeutic strategies.

Structural insights into DNA binding domain of vancomycin-resistance-associated response regulator in complex with its promoter DNA from Staphylococcus aureus.

In Staphylococcus aureus, vancomycin-resistance-associated response regulator (VraR) is a part of the VraSR two-component system, which is responsible for activating a cell wall-stress stimulon in response to an antibiotic that inhibits cell wall formation. Two VraR-binding sites have been identified: R1 and R2 in the vraSR operon control region. However, the binding of VraR to a promoter DNA enhancing downstream gene expression remains unclear. VraR contains a conserved N-terminal receiver domain (VraRN ) connected to a C-terminal DNA binding domain (VraRC ) with a flexible linker. Here, we present the crystal structure of VraRC alone and in complex with R1-DNA in 1.87- and 2.0-Å resolution, respectively. VraRC consisting of four α-helices forms a dimer when interacting with R1-DNA. In the VraRC -DNA complex structure, Mg2+ ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg2+ to VraRC enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraRC with R1- and R2-DNA revealed the essential residues that might play a crucial role in interacting with DNA of the vraSR operon. The structural information could help in designing and screening potential therapeutics/inhibitors to deal with antibiotic-resistant S. aureus via targeting VraR.

NMR structural analysis of DNA recognition by a novel Myb1 DNA-binding domain in the protozoan parasite Trichomonas vaginalis.

The transcription regulator, tvMyb1, is the first Myb family protein identified in Trichomonas vaginalis. Using an electrophoretic mobility shift assay, we defined the amino-acid sequence from Lys(35) to Ser(141) (tvMyb1(35-141)) as the minimal DNA-binding domain, encompassing two Myb-like DNA-binding motifs (designated as R2 and R3 motifs) and an extension of 10 residues at the C-terminus. NMR solution structures of tvMyb1(35-141) show that both the R2 and R3 motifs adopt helix-turn-helix conformations while helix 6 in the R3 motif is longer than its counterpart in vertebrate Myb proteins. The extension of helix 6 was then shown to play an important role in protein stability as well as in DNA-binding activity. The structural basis for the tvMyb1(35-141)/DNA interaction was investigated using chemical shift perturbations, residual dipolar couplings, DNA specificity data and data-driven macromolecular docking by HADDOCK. Our data indicate that the orientation between R2 and R3 motifs dramatically changes upon binding to DNA so as to recognize the DNA major groove through a number of key contacts involving residues in helices 3 and 6. The tvMyb1(35-141)/DNA complex model furthers our understanding of DNA recognition by Myb proteins and this approach could be applied in determining the complex structures involving proteins with multiple domains.

NMR assignments of vaccinia virus protein A28: an entry-fusion complex component.

Vaccinia virus (VACV) belonging to the poxvirus family enters the host cell via two different entry pathways; either endocytosis or virus/host cell membrane fusion. With respect to the virus/host cell membrane fusion, there are eleven viral membrane proteins forming a complicated entry-fusion complex (EFC), including A28, A21, A16, F9, G9, G3, H2, J5, L5, L1 and O3, to conduct the fusion function. These EFC components are highly conserved in all poxviruses and each of them is essential and necessary for the fusion activity. So far, with the exceptions of L1 and F9 whose crystal structures were reported, the structural information about other EFC components remains largely unclear. We aim to conduct a structural and functional investigation of VACV virus-entry membrane protein A28. In this work, we expressed and purified a truncated form of A28 (14 kDa; residues 38-146, abbreviated as tA28 hereinafter), with deletion of its transmembrane domain (residues 1-22) and a hydrophobic segment (residues 23-37). And the assignments of its backbone and side chain 1H, 13C and 15N chemical shifts of tA28 are reported. The secondary structure propensity from TALOS+ indicates that tA28 does contain three α-helices, six ß-strands and connecting loops. Aside from this, we demonstrated that tA28 does interact with fusion suppressor viral protein A26 (residues 351-500) by the 1H-15N HSQC spectrum. We interpret that A28 binding to A26 deactivates EFC fusion activity. The current study provides a valuable framework towards further structural analyses of this protein and for better understanding virus/host cell membrane fusion mechanism in association with virus entry.

DMTMM-Mediated Intramolecular Cyclization of Acidic Residues in Peptides/Proteins.

The formation of succinimide in proteins has attracted considerable attention in protein aging and biopharmaceutical research. The succinimide formation occurs spontaneously in proteins and is prone to hydrolysis to yield aspartate and isoaspartate, resulting in altered protein functions. Herein, we demonstrated that the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) can mediate intramolecular cyclization of aspartic acid to form succinimide efficiently in the LL37-derived short antimicrobial peptide KR12. The formation of succinimide in KR12 was confirmed by liquid chromatography tandem mass spectrometry and nuclear magnetic resonance. Moreover, the succinimide-containing KR12 displayed decreased antimicrobial activity, helicity, and serum stability in comparison with unmodified KR12. The succinimide formation usually changes the protein structure and function, and only in rare cases, it can help to maintain the protein stability. In addition to succinimide, DMTMM can also mediate intraresidue cyclization of N-terminal glutamate to form pyroglutamate. Our work thus provides a convenient and efficient method for preparation of succinimide/pyroglutamate-containing peptides, which can be used for studying their impact on peptide/protein function.

Solution structure and phospho-PmrA recognition mode of PmrD from Klebsiella pneumoniae.

In bacteria, the two-component system (TCS) is the most prevalent for sensing and transducing the environmental signals into the cell. In Salmonella, the small basic protein PmrD is found to protect phospho-PmrA and prolong the expression of PmrA-activated genes. In contrast, Escherichia coli PmrD fails to protect phospho-PmrA. Here, we show that Klebsiella pneumoniae PmrD (KP-PmrD) can inhibit the dephosphrylation of phospho-PmrA, and the interaction between KP-PmrD and the N-terminal receiver domain of PmrA (PmrA(N)) is much stronger in the presence than in the absence of the phosphoryl analog beryllofluoride (BeF(3)(-)) (K(D)=1.74 ± 0.81 µM vs. K(D)=236 ± 48 µM). To better understand the molecular interactions involved, the solution structure of KP-PmrD was found to comprise six ß-strands and a flexible C-terminal α-helix. Amide chemical shift perturbations of KP-PmrD in complex with BeF(3)(-)-activated PmrA(N) suggested that KP-PmrD may undergo a certain conformational rearrangement on binding to activated PmrA(N). Saturation transfer experiments revealed the binding surface to be located on one face of the ß-barrel. This finding was further verified by in vivo polymyxin B susceptibility assay of the mutants of KP-PmrD. The phospho-PmrA recognition surface of KP-PmrD, which involves two KP-PmrD proteins in complex with an activated-PmrA(N) dimer, is suggested to be a contiguous patch consisting of Trp3, Trp4, Ser23, Leu26, Glu27, Met28, Thr46, Leu48, Ala49, Asp50, Ala51, Arg52, Ile65, Asn67, Ala68, Thr69, His70, Tyr71, Ser73 and Glu74. Our study furthers the understanding of how PmrD protects phopho-PmrA in the PmrAB TCS.

Structure and DNA binding characteristics of the three-Cys(2)His(2) domain of mouse testis zinc finger protein.

The C-terminal three-Cys(2)His(2) zinc-finger domain (TZD) of mouse testis zinc-finger protein binds to the 5'-TGTACAGTGT-3' at the Aie1 (aurora-C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K(d) value of approximately 10(-8) M was obtained from surface plasmon resonance analysis for the TZD-DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical beta beta alpha fold. On binding to DNA, chemical shift perturbations and the R(2) transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5'-ATATGTACAGTGTTAT-3', are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence-specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions -1, 2, 3, and 6 of alpha-helices in fingers 1 and 2. The DNA sequence and nonsequence-specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein-DNA recognition.

N-Terminal Segment of TvCyP2 Cyclophilin from Trichomonas vaginalis Is Involved in Self-Association, Membrane Interaction, and Subcellular Localization.

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.

Insights Into Dynamics of Inhibitor and Ubiquitin-Like Protein Binding in SARS-CoV-2 Papain-Like Protease.

Covid-19 is caused by a novel form of coronavirus for which there are currently no vaccines or anti-viral drugs. This virus, termed SARS-CoV-2 (CoV2), contains Papain-like protease (PLpro) involved in viral replication and immune response evasion. Drugs targeting this protease therefore have great potential for inhibiting the virus, and have proven successful in older coronaviruses. Here, we introduce two effective inhibitors of SARS-CoV-1 (CoV1) and MERS-CoV to assess their potential for inhibiting CoV2 PLpro. We ran 1 µs molecular dynamics (MD) simulations of CoV2, CoV1, and MERS-CoV ligand-free PLpro to characterize the dynamics of CoV2 PLpro, and made comparisons between the three to elucidate important similarities and differences relevant to drug design and ubiquitin-like protein binding for deubiquitinating and deISGylating activity of CoV2. Next, we simulated the inhibitors bound to CoV1 and CoV2 PLpro in various poses and at different known binding sites to analyze their binding modes. We found that the naphthalene-based ligand shows strong potential as an inhibitor of CoV2 PLpro by binding at the putative naphthalene inhibitor binding site in both computational predictions and experimental assays. Our modeling work suggested strategies to improve naphthalene-based compounds, and our results from molecular docking showed that the newly designed compounds exhibited improved binding affinity. The other ligand, chemotherapy drug 6-mercaptopurine (6MP), showed little to no stable intermolecular interaction with PLpro and quickly dissociated or remained highly mobile. We demonstrate multiple ways to improve the binding affinity of the naphthalene-based inhibitor scaffold by engaging new residues in the unused space of the binding site. Analysis of CoV2 PLpro also brings insights into recognition of ubiquitin-like proteins that may alter innate immune response.

NMR assignments of protrusion domain of capsid protein from dragon grouper nervous necrosis virus.

Nervous necrosis virus (NNV) is a non-enveloped virus that causes massive mortality in aquaculture fish production worldwide. Recently X-ray crystallography and single particle cryo-EM have independently determined the icosahedral capsid of NNV to near-atomic resolutions to show the capsid protein is composed of a S-domain (shell) and a P-domain (protrusion) connected by a linker. However, the structure of the spike on NNV capsid made of trimeric P-domains was poorly resolved by cryo-EM. In addition, comparing the spike in the cryo-EM with that by X-ray suggests that the P-domain can move drastically relative to the shell, implicating an underlying structural mechanism during the infectious process. Yet, it remains unclear that such structural re-arrangement is ascribed to the change of the conformation of individual P-domain or in the association among P-domains. Given that molecular structure of the P-domain in solution phase is still lacking, we aim to determine the structure of the P-domain by solution NMR spectroscopy. In this communication, we report backbone and side chain 1H, 13C and 15N chemical shifts of the P-domain (residues 221-338) together with the linker region (residues 214-220), revealing ten ß-strands via chemical shift propensity analysis. Our findings are consistent with the X-ray crystal structure of the P-domain reported elsewhere. The current study provides a framework towards further structural analyses of the P-domain in various solution conditions.

1H, 13C and 15N resonance assignments and secondary structures of cyclophilin 2 from Trichomonas vaginalis.

Cyclophilins are peptidyl prolyl isomerases that play an important role in a wide variety of biological functions like protein folding and trafficking, intracellular and extracellular signaling pathways, nuclear translocation and in pre-mRNA splicing. Two cyclophilins have been identified in the parasitic organism Trichomonas vaginalis and were named as TvCyP1 and TvCyP2. The 2 enzymes have been found to interact with Myb transcription factors in the parasite which regulate the iron induced expression of ap65-1 gene leading to cytoadherence of the parasite to human vaginal epithelial cells to cause the disease trichomoniasis. TvCyP2 was found to interact specifically with Myb3 to regulate nuclear translocation of the transcription factor. It would be intriguing to identify the binding site of both proteins as it could pave way to newer targets for drug discovery. Here we report the 1H, 13C and 15N resonance assignments and secondary structure information of TvCyP2 that could help us investigate the interaction between Myb3 and TvCyP2 in detail using NMR.

Structural basis of interaction between dimeric cyclophilin 1 and Myb1 transcription factor in Trichomonas vaginalis.

Cyclophilin 1 (TvCyP1), a cyclophilin type peptidyl-prolyl isomerase present in the human parasite Trichomonas vaginalis, interacts with Myb1 and assists in its nuclear translocation. Myb1 regulates the expression of ap65-1 gene that encodes for a disease causing cytoadherence enzyme. Here, we determined the crystal structures of TvCyP1 and its complex with the minimum TvCyP1-binding sequence of Myb1 (Myb1104-111), where TvCyP1 formed a homodimer, unlike other single domain cyclophilins. In the complex structure, one Myb1104-111 peptide was bound to each TvCyP1 protomer, with G106-P107 and Y105 fitting well into the active site and auxiliary S2 pocket, respectively. NMR data further showed that TvCyP1 can catalyze the cis/trans isomerization of P107 in Myb1104-111. Interestingly, in the well-folded Myb1 protein (Myb135-141), the minimum binding sequence adopted a different conformation from that of unstructured Myb1104-111 peptide, that could make P107 binding to the active site of TvCyP1 difficult. However, NMR studies showed that similar to Myb1104-111 peptide, Myb135-141 also interacted with the active site of TvCyP1 and the dynamics of the Myb135-141 residues near P107 was reduced upon interaction. Together, the structure of TvCyP1 and detailed structural insights on TvCyP1-Myb1 interaction provided here could pave the way for newer drugs to treat drug-resistant strains.