Monothiol and dithiol glutaredoxin-1 from Clostridium oremlandii: identification of domain-swapped structures by NMR, X-ray crystallography and HDX mass spectrometry.

Protein dimerization or oligomerization resulting from swapping part of the protein between neighboring polypeptide chains is known to play a key role in the regulation of protein function and in the formation of protein aggregates. Glutaredoxin-1 from Clostridium oremlandii (cGrx1) was used as a model to explore the formation of multiple domain-swapped conformations, which were made possible by modulating several hinge-loop residues that can form a pivot for domain swapping. Specifically, two alternative domain-swapped structures were generated and analyzed using nuclear magnetic resonance (NMR), X-ray crystallography, circular-dichroism spectroscopy and hydrogen/deuterium-exchange (HDX) mass spectrometry. The first domain-swapped structure (ß3-swap) was formed by the hexameric cGrx1-cMsrA complex. The second domain-swapped structure (ß1-swap) was formed by monothiol cGrx1 (C16S) alone. In summary, the first domain-swapped structure of an oxidoreductase in a hetero-oligomeric complex is presented. In particular, a single point mutation of a key cysteine residue to serine led to the formation of an intramolecular disulfide bond, as opposed to an intermolecular disulfide bond, and resulted in modulation of the underlying free-energy landscape of protein oligomerization.

Interaction between human angiogenin and the p53 TAD2 domain and its implication for inhibitor discovery.

Interaction between angiogenin and the p53 TAD2 domain in cancer cells can inhibit the function of the p53 tumor suppressor and promote cell survival. Based on a model structure using NMR and mutational analysis, positively charged 31 RRR33 and 50 KRSIK54 motifs of human angiogenin were identified as p53-binding sites that could interact with negatively charged D48/E51 and E56 residues of the p53 TAD2 domain, respectively. These results suggest that 31 RRR33 and 50 KRSIK54 motifs of human angiogenin might play a critical role in the regulation of p53-mediated apoptosis and angiogenesis in cancer cells. This study identifies potential target sites for screening angiogenin-specific inhibitors that could not only inhibit p53 binding but could also simultaneously inhibit cell binding, internalization, DNA binding, and nuclear translocation of human angiogenin.

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.

Crystal structure of the EnvZ periplasmic domain with CHAPS.

Bacteria sense and respond to osmolarity through the EnvZ-OmpR two-component system. The structure of the periplasmic sensor domain of EnvZ (EnvZ-PD) is not available yet. Here, we present the crystal structure of EnvZ-PD in the presence of CHAPS detergent. The structure of EnvZ-PD shows similar folding topology to the PDC domains of PhoQ, DcuS, and CitA, but distinct orientations of helices and ß-hairpin structures. The CD and NMR spectra of EnvZ-PD in the presence of cholate, a major component of bile salts, are similar to those with CHAPS. Chemical cross-linking shows that the dimerization of EnvZ-PD is significantly inhibited by the CHAPS and cholate. Together with ß-galactosidase assay, these results suggest that bile salts may affect the EnvZ structure and function in Escherichia coli.

Structural Studies on the Extracellular Domain of Sensor Histidine Kinase YycG from Staphylococcus aureus and Its Functional Implications.

Bacterial two-component signal transduction systems are used to adapt to fluctuations in the environment. YycG, a key two-component histidine kinase in Staphylococcus aureus, plays an essential role in cell viability and regulates cell wall metabolism, biofilm formation, virulence, and antibiotic resistance. For these reasons, YycG is considered a compelling target for the development of novel antibiotics. However, to date, the signaling mechanism of YycG and its stimulus are poorly understood mainly because of a lack of structural information on YycG. To address this deficiency, we determined the crystal structure of the extracellular domain of S. aureus YycG (YycGex) at 2.0-Å resolution. The crystal structure indicated two subunits with an extracellular Per-Arnt-Sim (PAS) topology packed into a dimer with interloop interactions. Disulfide scanning using cysteine-substituted mutants revealed that YycGex possessed dimeric interfaces not only in the loop but also in the helix α1. Cross-linking studies using intact YycG demonstrated that it was capable of forming high molecular weight oligomers on the cell membrane. Furthermore, we also observed that two auxiliary proteins of YycG, YycH and YycI, cooperatively interfered with the multimerization of YycG. From these results, we propose that signaling through YycG is regulated by multimerization and binding of YycH and YycI. These structural studies, combined with biochemical analyses, provide a better understanding of the signaling mechanism of YycG, which is necessary for developing novel antibacterial drugs targeting S. aureus.

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.

Solid-state 31P NMR investigation on the status of guanine nucleotides in paclitaxel-stabilized microtubules.

Microtubule dynamics is a target for many chemotherapeutic drugs. In order to understand the biochemical effects of paclitaxel on the GTPase activity of tubulin, the status of guanine nucleotides in microtubules was investigated by (31)P cross-polarization magic angle spinning (CPMAS) NMR. Microtubules were freshly prepared in vitro in the presence of paclitaxel and then lyophilized in sucrose buffer for solid-state NMR experiments. A (31)P CPMAS NMR spectrum with the SNR of 25 was successfully acquired from the lyophilized microtubule sample. The broadness of the (31)P spectral lines in the spectrum indicates that the molecular environments around the guanine nucleotides inside tubulin may not be as crystalline as reported by many diffraction studies. Deconvolution of the spectrum into four spectral components was carried out in comparison with the (31)P NMR spectra obtained from five control samples. The spectral analysis suggested that about 13% of the nucleotides were present as GTP and 37% as GDP in the ß-tubulin (E-site) of the microtubules. It was found that most of the GDPs were present as GDP-Pi complex in the microtubules, which seems to be one of the effects of paclitaxel binding.

Mechanism of the pH-induced conformational change in the sensor domain of the DraK Histidine kinase via the E83, E105, and E107 residues.

The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A) is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces.

The dual binding site of angiogenin and its inhibition mechanism: the crystal structure of the rat angiogenin-heparin complex.

The heparin complex of rat angiogenin revealed that a heparin strand is fitted into a positively charged groove formed by the dual binding site of rat angiogenin, suggesting that cell adhesion to angiogenin is facilitated by its interaction with substrates on the cell surface and can be inhibited by heparin.

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.

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°.

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.

(1)H, (13)C, (15)N backbone and side-chain resonance assignments of rat angiogenin.

Angiogenin is an unusual member of the pancreatic ribonuclease superfamily that induces formation of new blood vessels and is a promising anti-cancer target. Here we report backbone and side chain (1)H, (13)C, and (15)N resonance assignments for rat angiogenin (residues 24-145), excluding the N-terminal signal peptide. These data allow nuclear magnetic resonance structure and inhibitor-binding studies with the aim of providing angiogenin antagonists as potential therapeutics.

Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane.

The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C-terminal periplasmic domain of OmpA (OmpA-like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA-like domains. To elucidate this molecular recognition process, we solved the high-resolution crystal structure of an OmpA-like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR-based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram-negative bacteria takes place in a DAP-dependent manner.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii.

Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal ß-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2(1), with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, ß = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å(3) Da(-1).

Crystal structure of the human histone methyltransferase ASH1L catalytic domain and its implications for the regulatory mechanism.

Absent, small, or homeotic disc1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addition, we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.

Rapid exploration of the folding topology of helical membrane proteins using paramagnetic perturbation.

An understanding of the folding states of α-helical membrane proteins in detergent systems is important for functional and structural studies of these proteins. Here, we present a rapid and simple method for identification of the folding topology and assembly of transmembrane helices using paramagnetic perturbation in nuclear magnetic resonance spectroscopy. By monitoring the perturbation of signals from glycine residues located at specific sites, the folding topology and the assembly of transmembrane helices of membrane proteins were easily identified without time-consuming backbone assignment. This method is validated with Mistic (membrane-integrating sequence for translation of integral membrane protein constructs) of known structure as a reference protein. The folding topologies of two bacterial histidine kinase membrane proteins (SCO3062 and YbdK) were investigated by this method in dodecyl phosphocholine (DPC) micelles. Combing with analytical ultracentrifugation, we identified that the transmembrane domain of YbdK is present as a parallel dimer in DPC micelle. In contrast, the interaction of transmembrane domain of SCO3062 is not maintained in DPC micelle due to disruption of native structure of the periplasmic domain by DPC micelle.

Structural characterization of the intra-membrane histidine kinase YbdK from Bacillus subtilis in DPC micelles.

Bacterial histidine kinases (HKs) play a critical role in signal transduction for cellular adaptation to environmental conditions and stresses. YbdK from Bacillus subtilis is a 320-residue intra-membrane sensing HK characterized by a short input domain consisting of two transmembrane helices without an extracytoplasmic domain. While the cytoplasmic domains of HKs have been studied in detail, the intra-membrane sensing domain systems are still uncharacterized due to difficulties in handling the transmembrane domain. Here, we successfully obtained pure recombinant transmembrane domain of YbdK (YbdK-TM) from E. coli and analyzed the characteristics of YbdK-TM using nuclear magnetic resonance (NMR) and other biophysical methods. YbdK-TM was found to form homo-dimers in DPC micelles based on cross-linking assays and analytical ultracentrifugation analyses. We estimated the size of the YbdK-TM DPC complex to be 46kDa using solution state NMR T(1)/T(2) relaxation analyses in DPC micelles. These results provide information that will allow functional and structural studies of intra-membrane sensing HKs to begin.

Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies.

Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D (15)N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly alpha-helical. This method should be applicable to the NMR analysis of other transmembrane proteins.

A new methodology for monitoring the activity of cdMMP-12 anchored and freeze-dried on Au (111).

Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state-based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors.