Search

PmrA, an OmpR/PhoB-family response regulator, activates gene transcription responsible for polymyxin resistance in bacteria by recognizing promoters in which the canonical -35 element is replaced by the pmra-box, representing the PmrA recognition sequence. Here, we report a cryo-electron microscopy-derived 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 substitution of PmrA DNA-recognition residues eliminated its transcriptional activity, but variants with altered RNAPH-interacting residues exhibited elevated transcriptional activity. Our study indicates that both PmrA recognition-induced DNA distortion and PmrA promoter escape play important roles in its transcriptional activation.

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 (VraR(N)) connected to a C‐terminal DNA binding domain (VraR(C)) with a flexible linker. Here, we present the crystal structure of VraR(C) alone and in complex with R1‐DNA in 1.87‐ and 2.0‐Å resolution, respectively. VraR(C) consisting of four α‐helices forms a dimer when interacting with R1‐DNA. In the VraR(C)–DNA complex structure, Mg(2+) ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg(2+) to VraR(C) enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraR(C) 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.

In Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg75 in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.

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 β-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 β-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.

The PhoQ/PhoP two-component system regulates the physiological and virulence functions of Salmonella enterica. However, the mode of action of known PhoP inhibitors is unclear. We systematically constructed a pharmacophore model of inhibitors to probe the interface pharmacophore model of the PhoP dimer, coupling it with Ligplot analysis. We found that these inhibitors bind on the α5-helix, altering the conformation and interfering with PhoP binding on DNA.

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.

The PhoQ/PhoP two-component system regulates the physiological and virulence functions of

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 (σ

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.

A new antibacterial drug is urgently needed. We employed a protein-DNA complex-guided pharmacophore modeling approach to screen inhibitors against the response regulator PmrA of polymyxin B-resistant Klebsiella pneumoniae (KP). The identified lead, E1 (IC50 = 10.2 μM), targeted the DNA-binding domain of PmrA (KD = 1.7 μM), whose conserved residues R171, R198, K203, and Y214 have been shown to be hotspots for antimicrobial development. Treatment of E1 restored the susceptibility of KP to polymyxin B.

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.

The mammalian ER protein STING (stimulators of interferon genes) is an important innate immunity protein for linking detection of novel secondary messengers c-di-GMP, c-di-AMP, cGAMP or cytosolic dsDNA to the activation of TANK kinase 1 and its downstream interferon regulator factor 3. Recently quite a few of crystal structures representing different states of the C-terminal domain (CTD) of human and murine STING (hSTING and mSTING) in complex with c-di-GMP, cGAMP or DMXAA have been reported. However, the C-terminal 42 residues of STING-CTD, which may be important in mediating the downstream reactions, is invisible or absent in all reported X-ray structures. In addition, X-ray crystal structures may be subject to crystal packing force. Hence an alternate method of determining the structure and function of STING in a near physiological condition is essential. We now report the near complete backbone resonance assignments of the 54 kDa dimeric mSTING-CTD in complex with DMXAA, which is the first step in determining its complex structure and understanding why DMXAA, which is a very efficient agent for curing mouse cancer, is totally ineffective in human.

Microbial infections of antibiotic-resistant strains cause serious diseases and have a significant impact on public health worldwide, so novel antimicrobial drugs are urgently needed. Insect venoms, a rich source of bioactive components containing antimicrobial peptides (AMPs), are attractive candidates for new therapeutic agents against microbes. Recently, a novel peptide, P1, identified from the venom of the Australian jumper ant Myrmecia pilosula, showed potent antimicrobial activities against both Gram-negative and Gram-positive bacteria, but its structure–function relationship is unknown. Here, we used biochemical and biophysical techniques coupled with computational simulations to explore the mode of action of P1 interaction with dodecylphosphocholine (DPC) micelles as a model membrane system. Our circular dichroism (CD) and NMR studies revealed an amphipathic α-helical structure for P1 upon interaction with DPC micelles. A paramagnetic relaxation enhancement approach revealed that P1 orients its α-helix segment (F6–G14) into DPC micelles. In addition, the α-helix segment could be essential for membrane permeabilization and antimicrobial activity. Moreover, the arginine residues R8, R11, and R15 significantly contribute to helix formation and membrane-binding affinity. The lysine residue K19 of the C-terminus functionally guides P1 to interact with DPC micelles in the early interaction stage. Our study provides insights into the mode of action of P1, which is valuable in modifying and developing potent AMPs as antibiotic drugs.

Previously, we identified that both fingers 1 and 2 in the three Cys2His2 zinc-finger domains (TZD) of testis zinc-finger protein specifically bind to its cognate DNA; however, finger 3 is non-sequence-specific. To gain insights into the interaction mechanism, here we further investigated the DNA-binding characteristics of TZD bound to non-specific DNAs and its finger segments bound to cognate DNA. TZD in non-specific DNA binding showed smaller chemical shift perturbations, as expected. However, the direction of shift perturbation, change of DNA imino-proton NMR signal, and dynamics on the 15N backbone atom significantly differed between specific and non-specific binding. Using these unique characteristics, we confirmed that the three single-finger segments (TZD1, TZD2 and TZD3) and the two-finger segment (TZD23) non-specifically bind to the cognate DNA. In comparison, the other two-finger segment (TZD12) binding to the cognate DNA features simultaneous non-specific and semi-specific binding, both slowly exchanged in terms of NMR timescale. The process of TZD binding to the cognate DNA is likely stepwise: initially TZD non-specifically binds to DNA, then fingers 1 and 2 insert cooperatively into the major groove of DNA by semi-specific binding, and finally finger 3 non-specifically binds to DNA, which promotes the specific binding on fingers 1 and 2 and stabilizes the formation of a specific TZD-DNA complex.

The transcription factor pituitary homeobox protein 2 (PITX2) is involved in genetic control of development. Mutations in PITX2, most in the homeodomain, cause the autosomal-dominant disorder Rieger syndrome. The mutants L16Q, K50E and R53P destabilize the structure and disrupt DNA-binding activity. The biological functions of these mutants have been characterized but not the structural basis behind the loss of DNA-binding activity. We performed multiple molecular dynamics simulations at 37°C to investigate the structural and dynamic effects of the 3 PITX2 homeodomain mutants. Compared with the wild type (WT), the L16Q mutant induces a kink in the α3 helix, which is stabilized by the hydrogen bond of Q21-R59. The disruption in backbone hydrogen bonds of V47-N51 and W48-R52 leads to a kink formation in the α3 helix of K50E. The R53P mutant alters the relative orientation of helices, which is apparently stabilized by the formation of new hydrogen bonds of T38-Q11, T38-Q12, T38-R2, N39-R2, L40-Q1, L40-R2, and T41-Q4. The hydrophobic core residues F8, L13, L40 and V45 change their positions in all mutants to break the hydrophobic core. Thus, changes in helical orientations and hydrophobic core cause rearrangement of the DNA-binding surface and disrupt DNA-binding activity in the mutants. The structural and molecular dynamics properties of 3 PITX2 homeodomain mutants differ from those of the WT, especially in formation of a kink in the recognition helix, change in the packing of helices and disruption of the hydrophobic core. This structural basis for the loss of DNA-binding activity for these polymorphisms may help in understanding the effect of mutations on other homeodomains with other diseases.

Chloroplasts protein precursors translated in the cytosol traverse the membranes to reach their intended destination with the help of translocon complexes called translocon at the outer envelope of chloroplasts and translocon at the inner envelope of chloroplasts (TIC), respectively. Two components of the TIC translocon, Tic110 and Tic40, which combine with Hsp93 (ClpC), are involved in protein translocation across the inner membrane into the stroma. The C-terminal NP-repeat domain of Tic40 (Tic40-NP) is homologous to the DP-repeat domain of co-chaperones Hsp70-interacting and Hsp70/Hsp90-organizing proteins. Interaction of Tic40-NP and Hsp93 stimulates ATP hydrolysis of Hsp93, but the hydrolysis is abolished in both N320A and N329A mutants of Tic40-NP. Here, we determined the nuclear magnetic resonance structure of Tic40-NP, which mainly consists of five α-helices stabilized by two hydrophobic cores. In addition, chemical shift perturbation results suggested that some residues at α1 and α5, as well as residues Asn320 and Asn329, cause conformational change on the two mutants, which may subsequently affect their binding to Hsp93. We provide valuable information for further investigating how Tic40-NP interacts with Hsp93.

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 β-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.

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.

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266-285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the self-assembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS alpha-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.

Ion channels are involved in diverse biological processes and play essential roles in the physiology of all cells. An increasing number of human and animal diseases have been identified as relating to the defective function of ion channels. Scorpion venoms contain various polypeptides with distinct biological functions that particularly affect the permeability of ion channels in cell membranes (Catterall 1980; Valdivia et al. 1992; Garcia et al. 1997). These polypeptides possess the potency to recognize ion channels and receptors in excitable membranes and are classified into four groups on the basis of ion-channel types: (1) group I modulates Na+-channel activity (Possani et al. 1999) and contains peptides of 60 to 70 amino acids linked by four disulfide bridges; (2) group II blocks K+ channels (Miller 1995; Romi-Lebrun et al. 1997) and are short peptides with 31 to 41 amino acid residues with three or four disulfide bonds; (3) group III supposedly inhibits Cl−channels (DeBin et al. 1993) and contains short-chain insect toxin peptides of ∼36 amino acids with four disulfide bonds; and (4) group IV includes peptides that modulate ryanodine-sensitive Ca2+ channels (Valdivia and Possani 1998). It is believed that the toxin has a unique tertiary structure that may provide valuable information for understanding channels. Thus, understanding the structural basis of the specificity of scorpion toxins for these receptors could lead to the design of new ligands with controlled activity and potency with potential for clinical applications. Scorpion K+-channel blockers of group II, named α-KTx, have been classified into 12 subfamilies (Miller 1995; Tytgat et al. 1999). These K+-channel blockers block two major classes of K+ channels: voltage-gated (Kv-type) and high-conductance Ca2+-activated (BK-type) K+ channels. The three-dimensional structures of several scorpion K+-channel blockers have been determined by NMR spectroscopy; these include charybdotoxin (ChTx; Bontems et al. 1991), iberiotoxin (IbTx; Johnson et al. 1992), noxiustoxin (NTx; Dauplais et al. 1995), PO5-NH2 (Meunier et al. 1993), kaliotoxin (KTx; Fernandez et al. 1994), margatoxin (MgTx; Johnson et al. 1994), and tityustoxin K-α (TsTx-Kα; Ellis et al. 2001). Although the overall fold of these α-KTx toxins is very similar, there are subtle variations among them in amino acid sequence, the size of the β-sheet, the type of β-turn, or the type of α-helix (i.e., α-helix versus 310-helix). These differences in toxin structure affect the placement of side-chain moieties. Thus, the selectivity that various scorpion toxins have for the outer vestibule of different K+ channels is typically quite distinct. Previously, Doyle et al. (1998) applied X-ray crystallographic methods to determine the three-dimensional structure of the KcsA bacterial K+ channel, which may serve as a good model for understanding the binding site of scorpion toxin on Kv-type channels. Recently, a new K+-channel blocker was identified from the scorpion venom of Tityus cambridgei (Tc1; Batista et al. 2000). Tc1 contains 23 amino acids linked with three disulfide bridges and is the smallest K+-channel blocker toxin from scorpion venoms. All previously known K+-channel blockers from scorpion venoms are longer than 30 amino acid residues and are classified into 12 subfamilies as described above. Tc1 is classified as the first member of the new subfamily 13. In K+-channel blocking activity, Tc1 recognizes the Shaker B K+ channels with a dissociation constant (Kd) of 65 nM and competes with NTx for binding to the synaptosomal membranes, with an inhibitory concentration 50% (IC50) value in the order of 200 nM (Batista et al. 2000). Tc1 is a highly basic peptide because it contains seven positively charged residues with a pI value of 9.50. The sequence alignment of Tc1 with eight other K+-channel blockers from scorpion toxins is shown in Figure 1 ▶. We found that six cysteine residues (Cys2, Cys5, Cys9, Cys15, Cys20, and Cys22), Gly13, and Lys14 (Tc1 numbering) are conserved, and the C-terminal regions are highly similar among these toxins. In addition, the sequence of Tc1 shows some unique properties. For example, Tc1 possesses Arg at position 19, whereas the corresponding residue in the other toxins is Lys. At position 16, Tc1 has Ile, whereas the other toxins, with the exception of the PO5 peptide, have Met at the corresponding position. Furthermore, Tc1 contains dense positively charged residues at residues 5–10. Unlike other scorpion toxins, Tc1 does not contain either negatively charged residues or proline. These properties make Tc1 an excellent candidate for three-dimensional structure determination and site-directed mutagenesis and for gaining clearer understanding of K+ channels. Fig. 1. Sequence alignment of Tc1 with eight other K+-channel blockers was generated using the CLUSTAL-W (Thompson et al. 1994) and ESPript (Gouet et al. 1999) programs. Three-dimensional solution structures for six of them have been determined: They are ChTx ... In this study, synthetic Tc1 was made by conventional solid-phase peptide synthesis and folded into its active conformation. We checked the channel-blocking activity of the synthesized Tc1 and found that both synthetic and native Tc1 possess similar blocking activity against the Shaker K+ channel. Next, we applied circular dichrosim (CD) and NMR techniques to solve the solution structure of Tc1. To further understand the various structure-function relationships among the K+-channel blockers from scorpion venoms, we compared the three-dimensional structure of Tc1 and those of other structurally and functionally related scorpion toxins, ChTx and NTx. We concluded that the C-terminal structure is the most important region for the blocking activity of Kv-type channels for scorpion K+-channel blockers. In addition, we also asserted that some of the residues in the larger scorpion K+-channel blockers, which contain 31 to 40 amino acids, are clearly not involved in K+-channel blocking activity.

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.

PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE–DNA complex structure revealed an REC–DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA–DNA complex, which reveals a similar yet different REC–DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC–DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC–DBD interdomain dynamics and the DBD–DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription., PmrA is a regulator of genes that are associated with antibiotic resistance. Here, the authors report the structure of the protein bound to DNA, and use NMR data to propose a mechanism for the regulation of gene transcription.

Cytotoxic ribonucleases found in the oocytes and early embryos of frogs with antitumor activity are well-documented. RC-RNase 2, a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana, consists of 105 residues linked with 4 disulfide bridges and belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Among the RC-RNases, the base preference for RNase 2 is UpG but CpG for RC-RNase 4; while RC-RNase possesses the base specificity of both UpG and CpG. Interestingly, RC-RNase 2 or 4 has much lower catalytic activity but only three-fold less cytotoxicity than RC-RNase. Here, we report the NMR solution structure of rRC-RNase 2, comprising three alpha-helices and two sets of antiparallel beta-sheets. The differences of side-chain conformations of subsite residues among RNase A, RC-RNase, RC-RNase 4 and rRNase 2 are related to their distinct catalytic activities and base preferences. Furthermore, the substrate-related residues in the base specificity among native RC-RNases are derived using the chemical shift perturbation on ligand binding.

Human pancreatitis-associated protein was identified in pathognomonic lesions of Alzheimer disease, a disease characterized by the presence of filamentous protein aggregates. Here, we showed that at physiological pH, human pancreatitis-associated protein forms non-Congo Red-binding, proteinase K-resistant fibrillar aggregates with diameters from 6 up to as large as 68 nm. Interestingly, circular dichroism and Fourier transform infrared spectra showed that, unlike typical amyloid fibrils, which have a cross-beta-sheet structure, these aggregates have a very similar secondary structure to that of the native protein, which is composed of two alpha-helices and eight beta-strands, as determined by NMR techniques. Surface structure analysis showed that the positively charged and negatively charged residues were clustered on opposite sides, and strong electrostatic interactions between molecules were therefore very likely, which was confirmed by cross-linking experiments. In addition, several hydrophobic residues were found to constitute a continuous hydrophobic surface. These results and protein aggregation prediction using the TANGO algorithm led us to synthesize peptide Thr(84) to Ser(116), which, very interestingly, was found to form amyloid-like fibrils with a cross-beta structure. Thus, our data suggested that human pancreatitis-associated protein fibrillization is initiated by protein aggregation primarily because of electrostatic interactions, and the loop from residues 84 to 116 may play an important role in the formation of fibrillar aggregates with a native-like conformation.

An enzymatic one-pot three-step glycosylation strategy was developed for the synthesis of sLex moiety of truncated PSGL-1 glycopeptide with and without sulfation. The method provided an efficient way to afford complex glycopeptides in a semi-preparative scale without further complicated and time-consuming purification process in each glycosylation step.

Indolicidin, a l3-residue antimicrobial peptide-amide, which is unusually rich in tryptophan and proline, is isolated from the cytoplasmic granules of bovine neutrophils. In this study, the structures of indolicidin in 50% D3-trifluoroethanol and in the absence and presence of SDS and D38-dodecylphosphocholine were determined using NMR spectroscopy. Multiple conformations were found and were shown to be due to different combinations of contact between the two WPW motifs. Although indolicidin is bactericidal and able to permeabilize bacterial membranes, it does not lead to cell wall lysis, showing that there is more than one mechanism of antimicrobial action. The structure of indolicidin in aqueous solution was a globular and amphipathic conformation, differing from the wedge shape adopted in lipid micelles, and these two structures were predicted to have different functions. Indolicidin, which is known to inhibit DNA synthesis and induce filamentation of bacteria, was shown to bind DNA in gel retardation and fluorescence quenching experiments. Further investigations using surface plasmon resonance confirmed the DNA-binding ability and showed the sequence preference of indolicidin. Based on our biophysical studies and previous results, we present a diagram illustrating the DNA-binding mechanism of the antimicrobial action of indolicidin and explaining the roles of the peptide when interacting with lipid bilayers at different concentrations.

A number of beta-microseminoproteins (MSPs) have been identified from different species. MSPs are all non-glycosylated and disulfide bond-rich, but show a relatively low level of conservation. Although all Cys residues are conserved, our previous study showed that the disulfide bond pairings differ in porcine and ostrich MSPs. Despite the variety of biological functions that have been suggested for MSPs, their real function is still poorly understood. Furthermore, no 3D structure has been reported for any MSP, so the determination of the structure and function of MSPs is an interesting and important task. In the present study, we determined the 3D solution structure of porcine MSP on the basis of 1018 restraints. The ensemble of 20 NMR structures was well defined, with average root-mean-square deviations of 0.83(+/-0.16) A for the backbone atoms and 1.37(+/-0.17) A for heavy-atoms in residues 2-90. The 3D structure showed that porcine MSP is clearly composed of two domains, an N-terminal domain consisting of one double-stranded and one four-stranded antiparallel beta-sheet, and a C-terminal domain consisting of two double-stranded antiparallel beta-sheet. The orientation of the two domains was derived mainly on the basis of long-range NOEs and verified using residual dipolar coupling data. No inter-domain hydrophobic interaction or H-bonding was detected. However, a number of charged residues were found in close proximity between the domains, indicating that electrostatic interaction may be the key factor for the orientation of the two domains. This is the first report of a 3D structure for any MSP. In addition, structural comparison based on distance matrix alignment (DALI), class architecture topology and homologous superfamily (CATH) and combinatorial extension (CE) methods revealed that porcine MSP has a novel structure with a new fold providing valuable information for future structural studies on other MSPs and for understanding their biological functions.

Serine proteases are among the most studied enzymes for their role as model enzymes for studying the enzyme catalytic mechanism and medical interest in their inhibition. We have applied NMR methods to determine the structure, dynamics, and catalytic mechanism of a serine protease, E. coli thioesterase/protease I (TEP-I). In this article we review the results of our efforts. We showed that TEP-I is an / / type SNGH-hydrolase with Ser10, Asp154 and His157 as the catalytic triad residues. In free TEP-I, His157 was found to forma strong hydrogen bond to Asp154, but not to Ser10-O H. Modelfree analysis of 15N-T1, 15N-T2 and 1H-15N NOE data revealed that TEP-I is a rigid protein with a flexible catalytic binding pocket. Slow motion involving segments around the catalytic site was detected. The formation of Michaelis complex (MC) between TEP-I and a transition state analogue, diethyl p-nitrophenyl phosphate (DENP), and its subsequent conversion to the tetrahedral complex (TC) follow a two-step process, a fast formation of MC followed by a slow conversion to TC. In both steps residues perturbed were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. From the large chemical shift perturbation upon conversion from MC to TC we proposed that the amide protons of Ser10 and Gly44 serve as the oxyanion-hole proton donors to stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP.

Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG). To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in alpha1 and Val96 in beta6, and participated in catalytic activity by hydrogen bonds to Lys9 and P(1) catalytic phosphate. Residues Thr35 and Asp67 determined B(1) base specificity, and Glu91 determined B(2) base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana . RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three α-helices and two sets of antiparallel β-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(±0.14) A for the backbone atoms, and 1.42(±0.19) A for the heavy atoms in residues 3–105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.

One of the critical intracellular signal transduction pathways involves the binding of the Grb2 SH2 domain to the phosphotyrosine (pTyr) motifs on growth factor receptors, such as epidermal growth factor receptor (EGFR) and erbB2, leading to downstream activation of the oncogenic Ras signaling pathway. Therefore, the Grb2 SH2 domain has been chosen as our target for the development of potential anticancer agents. As a continuation of our earlier work, herein we report the design and synthesis of new peptide analogs, and their inhibitory effect on the Grb2 SH2 domain using surface plasmon resonance (SPR) technology. These novel agents do not contain phosphotyrosine or phosphotyrosine mimics. Binding interactions between these peptides and the Grb2 SH2 domain were measured and analyzed using a BIAcore X instrument, which provides detailed information on the real-time detection of the binding interaction. The results of this study should provide important information for the further development of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain.

Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.

A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos.

Antimicrobial peptides are important components of the host innate defense mechanism against invading pathogens, especially for drug-resistant bacteria. In addition to bactericidal activity, the 25 residue peptide TP4 isolated from Nile tilapia also stimulates cell proliferation and regulates the innate immune system in mice. In this report, TP4 hyperpolarized and depolarized the membrane potential of Pseudomonas aeruginosa at sub-lethal and lethal concentrations. It also inhibited and eradicated biofilm formation. The in vitro binding of TP4 to bacterial outer membrane target protein, OprI, was markedly enhanced by a membrane-like surfactant sarkosyl and lipopolysaccharide, which converted TP4 into an α-helix. The solution structure of TP4 in dodecylphosphocholine was solved by NMR analyses. It contained a typical α-helix at residues Phe10-Arg22 and a distorted helical segment at Ile6-Phe10, as well as a hydrophobic core at the N-terminus and a cationic patch at the C-terminus. Residues Ile16, Leu19 and Ile20 in the hydrophobic face of the main helix were critical for the integrity of amphipathic structure, other hydrophobic residues played important roles in hemolytic and bactericidal activities. A model for the assembly of helical TP4 embedded in sarkosyl vesicle is proposed. This study may provide valuable insight for engineering AMPs to have potent bactericidal activity but low hemolytic activity.

In Trichomonas vaginalis, a Myb1 protein was previously demonstrated to repress transcription of an iron-inducible ap65-1 gene. In this study, a human cyclophilin A homologue, TvCyclophilin 1 (TvCyP1), was identified as a Myb1-binding protein using a bacterial two-hybrid library screening system. The recombinant TvCyP1 exhibited typical peptidyl-prolyl isomerase activity with kcat/Km of ∼7.1 μm(-1) s(-1). In a pulldown assay, the His-tagged Myb1 interacted with a GST-TvCyP1 fusion protein, which had an enzymatic proficiency half that of recombinant TvCyP1. Both the enzymatic proficiency of GST-TvCyP1 and its binding to His-Myb1 were eliminated by mutation of Arg(63) in the catalytic motif or inhibited by cyclosporin A. TvCyP1 was primarily localized to the hydrogenosomes by immunofluorescence assay, but it was also co-purified with Myb1 in certain vesicle fractions from differential and gradient centrifugations. Transgenic cells overexpressing HA-TvCyP1 had a higher level of nuclear Myb1 but a much lower level of Myb1 associated with the vesicles than control and those overexpressing HA-TvCyP1(R63A). Myb1 was detected at a much higher level in the HA-TvCyP1 protein complex than in the HA-TvCyP1(R63A) protein complex immunoprecipitated from P15 and P100, but not S100, fractions of postnuclear lysates. A TvCyP1-binding motif, (105)YGPKWNK(111), was identified in Myb1 in which Gly(106) and Pro(107) were essential for its binding to TvCyP1. Mutation of Gly(106) and Pro(107), respectively, in HA-Myb1 resulted in cytoplasmic retention and elevated nuclear translocation of the overexpressed protein. These results suggest that TvCyP1 may induce the release of Myb1 that is restrained to certain cytoplasmic vesicles prior to its nuclear translocation.

The small alpha-domain of Lon protease is thought to carry the substrate-recognition, nucleotide-binding, and DNA-binding sites. Here we report the complete resonance assignment of the alpha-domain for Bacillus subtilis Lon protease (Bs-Lon alpha-domain).

Escherichia coli thioesterase/protease I is a 183 amino acid protein with a molecular mass of 20500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a new GDSLS consensus motif, of which no structure has yet been determined. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytically active. From analysis of a set of heteronuclear multidimensional NMR spectra with uniform and specific amino acid labeled protein samples, we have obtained near-complete resonance assignments of the backbone 1H,13 C and 15N nuclei (BMRB databank accession number 4060). The secondary structure of E. coli thioesterase/protease I was further deduced from the consensus chemical shift indices, backbone short- and medium-range NOEs, and amide proton exchange rates. The protein was found to consist of four β-strands and seven α-helices, arranged in alternate order. The four β-strands were shown to form a parallel β-sheet. The topological arrangement of the β-strands of -1x, +2x, +1x appears to resemble that of the core region of the αβ hydrolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of β-strands and the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioesterase/protease I at present.

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 A. 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.

The solution conformation of a synthetic snake venom toxin waglerin I, has been determined by using proton nuclear magnetic resonance spectroscopy. By a combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of waglerin I was completely assigned. A set of 247 interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 2 dihedral angle restraints (from coupling constant measurements) and 7 omega torsion angel restraints for prolines, formed the basis of three-dimensional structure determined by molecular dynamics techniques. The 19 structures that were obtained satisfy the experimental restraints, and display small deviation from idealized covalent geometry. Analysis of converged structures indicates that the toxin has no special secondary structure. In the solution structure of waglerin I, the central ring region is well defined but the N- and C-termini possesses more disorder.

In Trichomonas vaginalis , a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

The mammalian ER protein STING (stimulator of interferon genes; also known as MITA, ERIS, MPYS or TMEM173) is an adaptor protein that links the detection of cytosolic dsDNA to the activation of TANK-binding kinase 1 (TBK1) and its downstream transcription factor interferon regulatory factor 3 (IFN3). Recently, STING itself has been found to be the direct receptor of bacterial c-di-GMP, and crystal structures of several human STING C-terminal domain (STING-CTD) dimers in the apo form or in complex with c-di-GMP have been published. Here, a novel set of structures of mouse STING-CTD (mSTING137-344) in apo and complex forms determined from crystals obtained under different crystallization conditions are reported. These novel closed-form structures exhibited considerable differences from previously reported open-form human STING-CTD structures. The novel mSTING structures feature extensive interactions between the two monomers, a unique asymmetric c-di-GMP molecule with one guanine base in an unusual syn conformation that is well accommodated in the dimeric interface with many direct specific interactions and two unexpected equivalent secondary peripheral c-di-GMP binding sites. Replacement of the amino acids crucial for specific c-di-GMP binding in mSTING significantly changes the ITC titration profiles and reduces the IFN-[beta] reporter luciferase activity. Taken together, these results reveal a more stable c-di-GMP binding mode of STING proteins that could serve as a template for rational drug design to stimulate interferon production by mammalian cells. Published version

NMR studies reveal that the conformational equilibrium of the HLDIIW fragment of endothelins, which is the C-terminal extension beyond the disulfide-linked core, is relatively little affected by changes in the structural preference within the bicyclic core. Rather the major determinant appears to be a hydrophobic interaction between the Trp ring and the lie19 sidechain. At subambient temperatures in aqueous medium, this interaction appears to be enhanced in species that have residues 12- 15 present and in a helical conformation.

Human regenerating (Reg) genes belong to the C-type lectin superfamily and express secretory proteins in various tissues. Reg Iα, also named lithostathine, has multiple roles in numerous biological events such as cytokines, anti-apoptotic factors and the calcium carbonate crystals inhibitor. Under physiological pH, Reg Iα becomes largely insoluble after a self-proteolysis process, and the N-terminally truncated form readily polymerizes into fibrils, which leads to neurodegenerative diseases. Reg Iα may form protofibril via lateral hydrophobic interactions with a native-like conformation. The structural basis from the native to fibril form, as well as the carbohydrate binding sites on Reg Iα, remain unknown. Here we present the NMR backbone and side-chain assignments of Reg Iα for use in further NMR investigations.