Pseudo-Isolated α-Helix Platform for the Recognition of Deep and Narrow Targets.

Although interest in stabilized α-helical peptides as next-generation therapeutics for modulating biomolecular interfaces is increasing, peptides have limited functionality and stability due to their small size. In comparison, α-helical ligands based on proteins can make steric clash with targets due to their large size. Here, we report the design of a monomeric pseudo-isolated α-helix (mPIH) system in which proteins behave as if they are peptides. The designed proteins contain α-helix ligands that do not require any covalent chemical modification, do not have frayed ends, and importantly can make sterically favorable interactions similar to isolated peptides. An optimal mPIH showed a more than 100-fold increase in target selectivity, which might be related to the advantages in conformational selection due to the absence of frayed ends. The α-helical ligand in the mPIH displayed high thermal stability well above human body temperature and showed reversible and rapid folding/unfolding transitions. Thus, mPIH can become a promising protein-based platform for developing stabilized α-helix pharmaceuticals.

Solution structure of the Z-DNA binding domain of PKR-like protein kinase from Carassius auratus and quantitative analyses of the intermediate complex during B-Z transition.

Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune response and viral infection. Structural and biophysical studies show that ZBPs initially form an intermediate complex with B-DNA for B-Z conversion. However, a comprehensive understanding of the mechanism of Z-DNA binding and B-Z transition is still lacking, due to the absence of structural information on the intermediate complex. Here, we report the solution structure of the Zα domain of the ZBP-containing protein kinase from Carassius auratus(caZαPKZ). We quantitatively determined the binding affinity of caZαPKZ for both B-DNA and Z-DNA and characterized its B-Z transition activity, which is modulated by varying the salt concentration. Our results suggest that the intermediate complex formed by caZαPKZ and B-DNA can be used as molecular ruler, to measure the degree to which DNA transitions to the Z isoform.

Ligand-Mediated Folding of the OmpA Periplasmic Domain from Acinetobacter baumannii.

The periplasmic domain of OmpA from Acinetobacter baumannii (AbOmpA-PD) binds to diaminopimelate and anchors the outer membrane to the peptidoglycan layer in the cell wall. Although the crystal structure of AbOmpA-PD with its ligands has been reported, the mechanism of ligand-mediated folding of AbOmpA remains elusive. Here, we report that in vitro refolded apo-AbOmpA-PD in the absence of ligand exists as a mixture of two partially folded forms in solution: mostly unfolded (apo-state I) and hololike (apo-state II) states. Binding of the diaminopimelate or glycine ligand induced complete folding of AbOmpA-PD. The apo-state I was highly flexible and contained some secondary structural elements, whereas the apo-state II closely resembled the holo-state in terms of both structure and backbone dynamics, except for the ligand-binding region. 15N-relaxation-dispersion analyses for apo-state II revealed substantial motion on a millisecond timescale of residues in the H3 helix near the ligand-binding site, with this motion disappearing upon ligand binding. These results provide an insight into the ligand-mediated folding mechanism of AbOmpA-PD in solution.

NMR elucidation of reduced B-Z transition activity of PKZ protein kinase at high NaCl concentration.

A Z-DNA binding protein (ZBP)-containing protein kinase (PKZ) in fish species has an important role in the innate immune response. Previous structural studies of the Zα domain of the PKZ from Carassius auratus (caZαPKZ) showed that the protein initially binds to B-DNA and induces B-Z transition of double stranded DNA in a salt concentration-dependent manner. However, the significantly reduced B-Z transition activity of caZαPKZ at high salt concentration was not fully understood. In this study, we present the binding affinity of the protein for B-DNA and Z-DNA and characterize its extremely low B-Z transition activity at 250 mM NaCl. Our results emphasize that the B-DNA-bound form of caZαPKZ can be used as molecular ruler to measure the degree of B-Z transition.

Differential ubiquitin binding by the acidic loops of Ube2g1 and Ube2r1 enzymes distinguishes their Lys-48-ubiquitylation activities.

The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184-196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1(1-183) (Ube2r1C). Replacement of Gln-105-Ser-106-Gly-107 in the acidic loop of Ube2r1C (Ube2r1C(YGY)) by the corresponding residues from Ube2g1 (Tyr-102-Gly-103-Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1C(C93S)-[(15)N]UB(K48R) oxyester displayed two-state conformational exchange, whereas the Ube2r1C(C93S/YGY)-[(15)N]UB(K48R) oxyester showed predominantly one state. Together with NMR studies that compared UB(K48R) oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.

Structural insight into the interaction between the Hox and HMGB1 and understanding of the HMGB1-enhancing effect of Hox-DNA binding.

The Hox DNA binding domain, the homeodomain, plays critical roles in genetic control of development and cell fate determination. The variable regulatory functions of Hox proteins are accomplished by binding to target DNA sequences and collaborating protein partners that includes human high mobility group B1 (HMGB1). To better understand the interaction between Hox and HMGB1 and the facilitation of Hox-DNA binding by HMGB1, we solved the solution structure of the homeodomain of Hox including the N-terminal arm region (Hoxc9DBD hereafter). In addition, the details of the interaction between these two proteins, as well as DNA binding of the Hox-HMGB1 complex, were investigated by NMR, ITC, and EMSA. The results suggest that binding of the HMGB1 A-box to Hoxc9DBD makes the loop-1 (loop preceding helix-2 of Hoxc9DBD) more access to DNA backbone, which facilitate Hox-DNA binding with enhanced affinity.

Characterization of the sensor domain of QseE histidine kinase from Escherichia coli.

In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6×His tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4-5.0, the folded protein existed in a monomeric form with a predominantly helical structure. (1)H-(15)N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE.

Template-Free Synthesis of a Molecular Solomon Link by Two-Component Self-Assembly.

A molecular Solomon link was synthesized in high yield through the template-free, coordination-driven self-assembly of a carbazole-functionalized donor and a tetracene-based dinuclear ruthenium(II) acceptor. The doubly interlocked topology was realized by a strategically chosen ligand which was capable of participating in multiple CH⋅⋅⋅π and π-π interactions, as evidenced from single-crystal X-ray analysis and computational studies. This method is the first example of a two-component self-assembly of a molecular Solomon link using a directional bonding approach. The donor alone was not responsible for the construction of the Solomon link, and was confirmed by its noncatenane self-assemblies obtained with other similar ruthenium(II) acceptors.

A high-affinity protein binder that blocks the IL-6/STAT3 signaling pathway effectively suppresses non-small cell lung cancer.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses for host defense and tumorigenic process. Upregulation of IL-6 is known to constitutively phosphorylate signal transducer and activator of transcription 3 (STAT3), leading to activation of multiple oncogene pathways and inflammatory cascade. Here, we present the development of a high-affinity protein binder, termed repebody, which effectively suppresses non-small cell lung cancer in vivo by blocking the IL-6/STAT3 signaling. We selected a repebody that prevents human IL-6 (hIL-6) from binding to its receptor by a competitive immunoassay, and modulated its binding affinity for hIL-6 up to a picomolar range by a modular approach that mimics the combinatorial assembly of diverse modules to form antigen-specific receptors in nature. The resulting repebody was highly specific for hIL-6, effectively inhibiting the STAT3 phosphorylation in a dose- and binding affinity-response manner in vitro. The repebody was shown to have a remarkable suppression effect on the growth of tumors and STAT3 phosphorylation in xenograft mice with non-small cell lung cancer by blocking the hIL-6/STAT3 signaling. Structural analysis of the repebody and IL-6 complex revealed that the repebody binds the site 2a of hIL-6, overlapping a number of epitope residues at site 2a with gp130, and consequently causes a steric hindrance to the formation of IL-6/IL-6Rα complex. Our results suggest that high-affinity repebody targeting the IL-6/STAT3 pathway can be developed as therapeutics for non-small cell lung cancer.

Design of a binding scaffold based on variable lymphocyte receptors of jawless vertebrates by module engineering.

Repeat proteins have recently been of great interest as potential alternatives to immunoglobulin antibodies due to their unique structural and biophysical features. We here present the development of a binding scaffold based on variable lymphocyte receptors, which are nonimmunoglobulin antibodies composed of Leucine-rich repeat modules in jawless vertebrates, by module engineering. A template scaffold was first constructed by joining consensus repeat modules between the N- and C-capping motifs of variable lymphocyte receptors. The N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach. The newly designed scaffold, termed "Repebody," showed a high level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering was shown by designing repebodies specific for myeloid differentiation protein-2 and hen egg lysozyme, respectively, by a rational approach. The crystal structures of designed repebodies were determined to elucidate the structural features and interaction interfaces. We demonstrate general applicability of the scaffold by selecting repebodies with different binding affinities for interleukin-6 using phage display.

Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway.

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1-RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1-RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432-Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1-RASSF5 complex showed a longer helical structure (Ser438-Lys480) than that in the MST1 homodimer (Val441-Lys480). Moreover, extensive polar and nonpolar contacts in the MST1-RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST-RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1-RASSF5 SARAH domain in apoptosis signalling.

Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells.

Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesion-dependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer.

Expression, purification and characterization of human vacuolar-type H(+)-ATPase subunit d1 and d2 in Escherichia coli.

Vacuolar-type H(+)-ATPase (V-ATPase) is a multi-subunit proton pump. The proton pump is essential for the regulation of pH in various eukaryotic cellular processes. Among the 14 subunits that constitute V-ATPase, d subunit mediates coupling between cytosolic and membrane domains. Whereas d1 is expressed ubiquitously in various types of cells, its isoform d2 is only expressed in specific cells or tissues. To characterize these isoforms, we expressed and purified the isoforms of human V-ATPase d subunits using Escherichia coli over-expression system. Subunit d1 and d2 were purified as homogeneous monomers as demonstrated by dynamic light scattering (DLS) analysis. Secondary structures of d subunits were estimated to be composed of 73% α-helix and 2% ß-sheet, as analyzed using circular dichroism (CD) analysis. Although sequence identity and secondary structures of d subunits were highly similar, the relative stability against thermal stress was higher for d1 than d2. Efficient expression and purification of d subunits, together with biophysical and biochemical characterization, presented in this study is expected to facilitate further structural analysis to clarify specific inter-molecular interactions involved in multi-subunit assembly and regulation of H(+) transporters.

Solution structure of the Zbeta domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs.

The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zα and Zß, and an adjacent putative B-DNA binding domain. The crystal structure of the Zß domain of human DAI (hZß(DAI)) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZß(DAI), the solution structure of the free hZß(DAI) was solved, and its bindings to B- and Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA-bound structure, the conformation of free hZß(DAI) has notable alterations in the α3 recognition helix, the "wing," and Y145, which are critical in Z-DNA recognition. Unlike some other Zα domains, hZß(DAI) appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZß(DAI) also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZß(DAI) is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs.

Crystal structures of the LsrR proteins complexed with phospho-AI-2 and two signal-interrupting analogues reveal distinct mechanisms for ligand recognition.

Quorum sensing (QS) is a cell-to-cell communication system responsible for a variety of bacterial phenotypes including virulence and biofilm formation. QS is mediated by small molecules, autoinducers (AIs), including AI-2 that is secreted by both Gram-positive and -negative microbes. LsrR is a key transcriptional regulator that governs the varied downstream processes by perceiving AI-2 signal, but its activation via autoinducer-binding remains poorly understood. Here, we provide detailed regulatory mechanism of LsrR from the crystal structures in complexes with the native signal (phospho-AI-2, D5P) and two quorum quenching antagonists (ribose-5-phosphate, R5P; phospho-isobutyl-AI-2, D8P). Interestingly, the bound D5P and D8P molecules are not the diketone forms but rather hydrated, and the hydrated moiety forms important H-bonds with the carboxylate of D243. The D5P-binding flipped out F124 of the binding pocket, and resulted in the disruption of the dimeric interface-1 by unfolding the α7 segment. However, the same movement of F124 by the D8P'-binding did not cause the unfolding of the α7 segment. Although the LsrR-binding affinity of R5P (Kd, ∼1 mM) is much lower than that of D5P and D8P (∼2.0 and ∼0.5 µM), the α-anomeric R5P molecule fits into the binding pocket without any structural perturbation, and thus stabilizes the LsrR tetramer. The binding of D5P, not D8P and R5P, disrupted the tetrameric structure and thus is able to activate LsrR. The detailed structural and mechanistic insights from this study could be useful for facilitating design of new antivirulence and antibiofilm agents based on LsrR.

Crystal structures of 26kDa Clonorchis sinensis glutathione S-transferase reveal zinc binding and putative metal binding.

The crystal structures of CsGST in two different space groups revealed that Asp26 and His79 coordinate a zinc ion. In one space group, His46 of an adjacent molecule participates in the coordination within 2.0Å. In the other space group, Asp26, His79 and a water molecule coordinate a zinc ion. The CsGST-D26H structure showed that four histidine residues - His26 and His79 from one molecule and the same residues from a symmetry-related neighboring molecule - coordinate a zinc ion. The coordinated zinc ions are located between two molecules and mediate molecular contacts within the crystal.

pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor.

Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5-10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.

Expression, purification, crystallization and preliminary X-ray analysis of the extracellular sensory domain of DraK histidine kinase from Streptomyces coelicolor.

The bacterium Streptomyces coelicolor produces useful antibiotics from its secondary metabolites. DraK is a sensory histidine kinase involved in the differential regulation of antibiotics in S. coelicolor through the DraR/DraK two-component system. Here, the extracellular sensory domain of DraK was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal diffracted to 2.2 Å resolution and belonged to space group C2221, with unit-cell parameters a = 41.91, b = 174.50, c = 145.25 Å, α = ß = γ = 90°.

Magnetic control of self-assembly and disassembly in organic materials.

Because organic molecules and materials are generally insensitive or weakly sensitive to magnetic fields, a certain means to enhance their magnetic responsiveness needs to be exploited. Here we show a strategy to amplify the magnetic responsiveness of self-assembled peptide nanostructures by synergistically combining the concepts of perfect α-helix and rod-coil supramolecular building blocks. Firstly, we develop a monomeric, nonpolar, and perfect α-helix (MNP-helix). Then, we employ the MNP-helix as the rod block of rod-coil amphiphiles (rod-coils) because rod-coils are well-suited for fabricating responsive assemblies. We show that the self-assembly processes of the designed rod-coils and disassembly of rod-coil/DNA complexes can be controlled in a magnetically responsive manner using the relatively weak magnetic field provided by the ordinary neodymium magnet [0.07 ~ 0.25 Tesla (T)]. These results demonstrate that magnetically responsive organic assemblies usable under practical conditions can be realized by using rod-coil supramolecular building blocks containing constructively organized diamagnetic moieties.

Expression, purification, crystallization and preliminary X-ray analysis of the human NORE1 SARAH domain.

NORE1 is an important tumour suppressor in human cancers that interacts with the pro-apoptotic protein kinase MST1/2 through SARAH domains. The SARAH domain (residues 366-413) of human NORE1 was expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 2.7 Šresolution and belonged to space group P6(1)22, with unit-cell parameters a = b = 73.041, c = 66.092 Å, α = ß = 90, γ = 120°.