Conformational Changes of α-Crystallin Proteins Induced by Heat Stress.

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.

Adaptation of thermophilic acetyltransferase to a water-mediated catalytic mechanism.

The common mechanism of N-acetyltransferases (NATs) is a water-mediated catalysis, which is not conducive to thermophilic acetyltransferases. The crystal structure of SsArd1 shows an ordered catalytic water molecule in a trap formed by the residues H88 and E127. Structure-guided mutagenesis, kinetic studies and MD simulation indicated that the turnover rates of H88A, E127A and H88A/E127A mutants were low, but that of the H88E/E127H mutant could be restored to the level of the wild type.

The crystal structure of protein-transporting chaperone BCP1 from Saccharomyces cerevisiae.

BCP1 is a protein enriched in the nucleus that is required for Mss4 nuclear export and identified as the chaperone of ribosomal protein Rpl23 in Saccharomyces cerevisiae. According to sequence homology, BCP1 is related to the mammalian BRCA2-interacting protein BCCIP and belongs to the BCIP protein family (PF13862) in the Pfam database. However, the BCIP family has no discernible similarity to proteins with known structure. Here, we report the crystal structure of BCP1, presenting an α/ß fold in which the central antiparallel ß-sheet is flanked by helices. Protein structural classification revealed that BCP1 has similarity to the GNAT superfamily but no conserved substrate-binding residues. Further modeling and protein-protein docking work provide a plausible model to explain the interaction between BCP1 and Rpl23. Our structural analysis presents the first structure of BCIP family and provides a foundation for understanding the molecular basis of BCP1 as a chaperone of Rpl23 for ribosome biosynthesis.

Crystal Structure of α-Galactosidase from Thermus thermophilus: Insight into Hexamer Assembly and Substrate Specificity.

α-Galactosidase catalyzes the hydrolysis of a terminal α-galactose residue in galacto-oligosaccharides and has potential in various industrial applications and food processing. We determined the crystal structures of α-galactosidase from the thermophilic microorganism Thermus thermophilus (TtGalA) and its complexes with pNPGal and stachyose. The monomer folds into an N-terminal domain, a catalytic (ß/α)8 barrel domain, and a C-terminal domain. The domain organization is similar to that of the other family of 36 α-galactosidases, but TtGalA presents a cagelike hexamer. Structural analysis shows that oligomerization may be a key factor for the thermal adaption of TtGalA. The structure of TtGalA complexed with stachyose reveals only the existence of one -1 subsite and one +1 subsite in the active site. Structural comparison of the stachyose-bound complexes of TtGalA and GsAgaA, a tetrameric enzyme with four subsites, suggests evolutionary divergence of substrate specificity within the GH36 family of α-galactosidases. To the best of our knowledge, the crystal structure of TtGalA is the first report of a quaternary structure as a hexameric assembly in the α-galactosidase family.

C-terminal Redox Domain of Arabidopsis APR1 is a Non-Canonical Thioredoxin Domain with Glutaredoxin Function.

Sulfur is an essential nutrient that can be converted into utilizable metabolic forms to produce sulfur-containing metabolites in plant. Adenosine 5'-phosphosulfate (APS) reductase (APR) plays a vital role in catalyzing the reduction of activated sulfate to sulfite, which requires glutathione. Previous studies have shown that the C-terminal domain of APR acts as a glutathione-dependent reductase. The crystal structure of the C-terminal redox domain of Arabidopsis APR1 (AtAPR1) shows a conserved α/ß thioredoxin fold, but not a glutaredoxin fold. Further biochemical studies of the redox domain from AtAPR1 provided evidence to support the structural observation. Collectively, our results provide structural and biochemical information to explain how the thioredoxin fold exerts the glutaredoxin function in APR.

Comparison of the Effects of Daptomycin on Bacterial and Model Membranes.

Daptomycin is a phosphatidylglycerol specific, calcium-dependent membrane-active antibiotic that has been approved for the treatment of Gram-positive infections. A recent Bacillus subtilis study found that daptomycin clustered into fluid lipid domains of bacterial membranes and the membrane binding was correlated with dislocation of peripheral membrane proteins and depolarization of membrane potential. In particular, the study disproved the existence of daptomycin ion channels. Our purpose here is to study how daptomycin interacts with lipid bilayers to understand the observed phenomena on bacterial membranes. We performed new types of experiments using aspirated giant vesicles with an ion leakage indicator, making comparisons between daptomycin and ionomycin, performing vesicle-vesicle transfers, and measuring daptomycin binding to fluid phase versus gel phase bilayers and bilayers including cholesterol. Our findings are entirely consistent with the observations for bacterial membranes. In addition, daptomycin is found to cause ion leakage through the membrane only if its concentration in the membrane is over a certain threshold. The ion leakage caused by daptomycin is transient. It occurs only when daptomycin binds the membrane for the first time; afterward, they cease to induce ion leakage. The ion leakage effect of daptomycin cannot be transferred from one membrane to another. The level of membrane binding of daptomycin is reduced in the gel phase versus the fluid phase. Cholesterol also weakens the membrane binding of daptomycin. The combination of membrane concentration threshold and differential binding is significant. This could be a reason why daptomycin discriminates between eukaryotic and prokaryotic cell membranes.

Molecular State of the Membrane-Active Antibiotic Daptomycin.

Membrane-active antibiotics are potential alternatives to the resistance-prone conventional antibiotics. Daptomycin, a cyclic lipopeptide, is the only membrane-active antibiotic approved by the U.S. Food and Drug Administration so far. The drug interacts with the cytoplasmic membranes of Gram-positive pathogens, causing membrane permeabilization to ions and cell death. The antibiotic activity is calcium-ion dependent and correlates with the target membrane's content of phosphatidylglycerol (PG). For such a complex reaction with membranes, it has been difficult to uncover the molecular process that underlies its antibacterial activity. The role of the cofactor, calcium ions, has been confusing. Many have proposed that calcium ions binding to daptomycin is a precondition for membrane interaction. Here, we report our findings on the molecular state of daptomycin before and after its membrane-binding reaction, particularly at therapeutic concentrations in the low micromolar range. We were able to perform small-angle x-ray scattering at sufficiently low daptomycin concentrations to determine that the molecules are monomeric before membrane binding. By careful circular dichroism (CD) analyses of daptomycin with Ca2+ and PG-containing membranes, we found that there are only two states identifiable by CD, one before and another after membrane binding; all other CD spectra are linear combinations of the two. Before membrane binding, the molecular state of daptomycin as defined by CD is the same with or without calcium ions. We are able to determine the stoichiometric ratios of the membrane-binding reaction. The stoichiometric ratio of daptomycin to calcium is 2:3. The stoichiometric ratio of daptomycin to PG is ∼1:1 if only the PG lipids in the outer leaflets of membranes are accessible to daptomycin.

Multiple Conformations of the Loop Region Confers Heat-Resistance on SsArd1, a Thermophilic NatA.

Structural comparison indicates that the loop region between ß3 and ß4 of SsArd1 is extended relative to the corresponding region in mesophilic Nats, and forms a plastic hydrogen-bond network mainly at two serine residues. Strikingly, two single-point mutants showed ∼3 °C decrease in melting temperature, and two other variants showed ∼7 °C decrease; this correlated with significantly reduced enzymatic activity. To our knowledge, this is the first discovery of a loop region capable of remarkably improving protein thermostability. This provides a novel route to engineer heat-resistant proteins.

Structural basis for substrate-specific acetylation of Nα-acetyltransferase Ard1 from Sulfolobus solfataricus.

Nα-acetyltransferases (Nats) possess a wide range of important biological functions. Their structures can vary according to the first two residues of their substrate. However, the mechanisms of substrate recognition and catalysis of Nats are elusive. Here, we present two structure of Sulfolobus solfataricus Ard1 (SsArd1), a member of the NatA family, at 2.13 and 1.84 Å. Both structures contain coenzyme A, while the latter also contains a substrate-derived peptide. Sequential structure-based mutagenesis revealed that mutations of critical residues for CoA binding decreased the binding affinity of SsArd1 by 3 ~ 7-fold. Superimposition of SsArd1 (NatA) with human Naa50p (NatE) showed significant differences in key residues of enzymes near the first amino-acid position of the substrate peptide (Glu35 for SsArd1 and Val29 for Naa50p). Further enzyme activity assays revealed that the substrate specificity of SsArd1 could be altered from SSGTPT to MEEKVG by a range of Glu35 mutants. These studies provide not only a molecular elucidation of substrate recognition and specificity for the NatA family, but also insight into how members of the NAT family distinguish between amino acids at the substrate N-terminus from the ancient monomeric archaeal Ard1.

Crystallization of the C-terminal redox domain of the sulfur-assimilatory enzyme APR1 from Arabidopsis thaliana.

Plant-type APS reductase (APR), which catalyzes the reduction of activated sulfate to sulfite in plants, consists of a reductase domain and a C-terminal redox domain showing sequence homology to thioredoxin but possessing the activity of glutaredoxin. In order to understand the structural and biochemical properties of the redox domain of plant-type APS reductase, the C-terminal domain of APR1 (APR1C) from Arabidopsis thaliana was crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to a resolution of 2.70 Šon the SPXF beamline BL13B1 at the NSRRC, Taiwan. The crystals belonged to space group P43212 or P41212, with unit-cell parameters a = b = 58.2, c = 86.7 Å. With one molecule per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.64 Å(3) Da(-1), which corresponds to a solvent content of approximately 53.49%. Further structure-based functional studies of APR1C would extend knowledge of the molecular mechanism and regulation of APR.

Structural basis for DNA-mediated allosteric regulation facilitated by the AAA+ module of Lon protease.

Lon belongs to a unique group of AAA+ proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease from Brevibacillus thermoruber (Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+ module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain-domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+ module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.

Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus.

Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni(2+)-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80 Šon the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.3, b = 86.1, c = 118.2 Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.52 Å(3) Da(-1) and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement.

Using single-molecule approaches to study archaeal DNA-binding protein Alba1.

Thermophilic and hyperthermophilic archaea have one or more copies of the Alba gene, which encodes Alba, a dimeric, highly basic protein that binds cooperatively to DNA. However, the functions of Alba and how it interacts with DNA remain unclear. In this study, we have used single-molecule tethered particle motion (TPM) and optical tweezers (OT) experiments to study the interactions between DNA molecules and Alba1. When Alba1 binds to double-stranded DNA, the Brownian motion (BM) amplitude for DNA tethers increases continuously, suggesting that Alba1 binds cooperatively. The OT study confirmed that a 5-fold increase in the persistence length of the Alba1 nucleoprotein filament is the major factor causing the increase in the BM amplitude for DNA tethers, while the contour length remained mostly unchanged. Moreover, the rate of the increase in the BM amplitude and the BM plateau value are both DNA length-dependent, indicating that the number of Alba1 initiation binding sites increases as the DNA becomes longer. Using the incoming-strand TPM experiment to monitor the interaction between Alba1 nucleoprotein filaments, we found that significant dimer-dimer contacts between two Alba1 nucleoprotein filaments are present, and the interaction is regulated by the concentration of Alba1.

Crystallization and preliminary X-ray diffraction analysis of the α subdomain of Lon protease from Brevibacillus thermoruber.

DNA-binding ability has previously been reported as a novel function for the thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon), and the α subdomain (amino acids 491-605) of Bt-Lon has been identified as being responsible for DNA binding. However, the physiological role and DNA-recognition mode of Bt-Lon still remain unclear. In this study, the crystallization and preliminary crystallographic analysis of the Bt-Lon α subdomain are presented. Native diffraction data to 2.88 Å resolution were obtained from a vitrified crystal at 100 K on the BL13C1 beamline at the NSRRC (National Synchrotron Radiation Research Center), Taiwan. The crystals belonged to space group P23, with unit-cell parameters a = b = c = 94.28 Å. Solvent-content calculations and molecular-replacement results suggest that there are two molecules of Bt-Lon α subdomain per asymmetric unit.

Identification and characterization of an extracellular alkaline phosphatase in the marine diatom Phaeodactylum tricornutum.

In phosphorus-deficient conditions, Phaeodactylum tricornutum releases an alkaline phosphatase (PtAPase) to the medium that is readily detectable by activity staining. Nucleic acid and amino acid sequence of this alkaline phosphatase (APase) was identified by performing proteomic analysis and database searches. Sequence alignment suggests that PtAPase belongs to the PhoA family, and it possesses key residues at the Escherichia coli PhoA active site. Quantitative PCR results indicate that the induction of APase mRNA transcription is very sensitive to phosphorus availability and population growth. The molecular mass of native PtAPase (148 kDa) determined by gel filtration chromatography indicates that PtAPase, like most PhoA, is homodimeric. Zn and Mg ions are essential cofactors for most PhoA enzymes; however, PtAPase activity did not require Zn ions. In fact, 5 mM Zn²âº, Mo²âº, Co²âº, Cd²âº, or Cu²âº inhibited its enzymatic activity, whereas 5 mM Mn²âº, Mg²âº, or Ca²âº enhanced its enzymatic activity. The responses of PtAPase to divalent metal ions were different from those of most PhoAs, but were similar to the PhoA in a marine bacterium, Cobetia marina. Phylogenetic analysis shows that homologs of PhoA are also present in other diatom species, and that they clustered in a unique branch away from other PhoA members. PtAPase may represent a novel class of PhoA that helps diatoms to survive in the ocean. Quantification of the PtAPase mRNA may help monitor the physiological condition of diatoms in natural environments and artificial bioreactors.

Crystal structures and molecular dynamics simulations of thermophilic malate dehydrogenase reveal critical loop motion for co-substrate binding.

Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60 °C and specific activity of about 2,600 U/mg with optimal temperature of 90 °C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme's activity.

Solution structure of the oncogenic MIEN1 protein reveals a thioredoxin-like fold with a redox-active motif.

The novel tumor biomarker MIEN1, identified by representational difference analysis, is overexpressed in breast cancer and prostate cancer. MIEN1 is considered an oncogenic protein, because MIEN1 overexpression functionally enhances migration and invasion of tumor cells via modulating the activity of AKT. However, the structure and molecular function of MIEN1 is little understood. Here, we report the solution structure of MIEN1, which adopts a thioredoxin-like fold with a redox-active motif. Comparison of backbone chemical shifts showed that most of the residues for both oxidized and reduced MIEN1 possessed the same backbone conformation, with differences limited to the active motif and regions in proximity. The redox potential of this disulfide bond was measured as -225 mV, which compares well with that of disulfides for other thioredoxin-like proteins. Overall, our results suggest that MIEN1 may have an important regulatory role in phosphorylation of AKT with its redox potential.

Resonance assignments of human C35 (C17orf37) protein, a novel tumor biomarker.

A new tumor-specific target, termed C35 (C17orf37), has been identified by representational difference analysis of tumor and normal human mammary cell lines. C35 protein is considered to be an important target for cancer therapy, since the over expression of C35 functionally enhances migration and invasion of tumor cells. Here we report the NMR resonance assignments of C35 protein for further structural determination and functional studies.

Preliminary crystallographic characterization of the Grb2 SH2 domain in complex with a FAK-derived phosphotyrosyl peptide.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein with a single SH2 domain that specifically binds to focal adhesion kinase (FAK) when residue Tyr925 of FAK is phosphorylated. The Grb2-FAK interaction is associated with cellular integrin-activated signal transduction events leading to the activation of the Ras-MAPK pathway. Crystals of the Grb2 SH2 domain in complex with a phosphopeptide corresponding to residues 921-930 of FAK have been obtained using the sitting-drop vapour-diffusion technique. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 102.7, c = 127.6 A, alpha = beta = 90.0, gamma = 120.0 degrees . A diffraction data set was collected from a flash-cooled crystal at 100 K to 2.49 A resolution using synchrotron radiation. Structure determination by molecular replacement and analysis of the detailed structure of the complex are currently in progress.