Higher Oligomeric States Research Articles

Structural and mechanistic insights into ALS patient derived mutations in D-amino acid oxidase

The D-amino acid oxidase protein modulates neurotransmission by controlling the levels of D-serine, a co-agonist of N-methyl-D-aspartate receptors. Mutations in the DAO gene have been associated with ALS, with some studies reporting pathogenic mechanisms of the R199W mutation. We have characterized two novel mutations R38H and Q201R found in ALS patients and report certain novel findings related to the R199W mutation. We report the first instance of crystal structure analysis of a patient-derived mutant of DAO, R38H, solved at 2.10 Å. The structure revealed significant perturbations and altered binding with the cofactor (FAD) and the inhibitor benzoate, supported by biochemical assays. Q201R-DAO also exhibited significantly lower ligand binding efficiency. Furthermore, kinetic analysis across all variants revealed reduced oxidase activity and substrate binding. Notably, R38H-DAO exhibited near-WT activity only at high substrate concentrations, while R199W-DAO and Q201R-DAO displayed drastic activity reduction. Additionally, structural perturbations were inferred for R199W-DAO and Q201R-DAO, evident by the higher oligomeric state in the holoenzyme form. We also observed thermal instability in case of R199W-DAO mutant. We hypothesize that the mutant enzymes may be rendered non-functional in a cellular context, potentially leading to NMDAR-associated excitotoxicity. The study provides novel insights into structural and functional aspects of DAO mutations in ALS.

The Molecular Assembly State of Drp1 Controls its Association With the Mitochondrial Recruitment Receptors Mff and MIEF1/2

Drp1 is a central player in mitochondrial fission and is recruited to mitochondria by Mff and MIEFs (MIEF1 and MIEF2), but little is known about how its assembly state affects Drp1 mitochondrial recruitment and fission. Here, we used in vivo chemical crosslinking to explore the self-assembly state of Drp1 and how it regulates the association of Drp1 with MIEFs and Mff. We show that in intact mammalian cells Drp1 exists as a mixture of multiple self-assembly forms ranging from the minimal, probably tetrameric, self-assembly subunit to several higher order oligomers. Precluding mitochondria-bound Drp1 in Mff/MIEF1/2-deficient cells does not affect the oligomerization state of Drp1, while conversely forced recruitment of Drp1 to mitochondria by MIEFs or Mff facilitates Drp1 oligomerization. Mff preferentially binds to higher order oligomers of Drp1, whereas MIEFs bind to a wider-range of Drp1 assembly subunits, including both lower and higher oligomeric states. Mff only recruits active forms of Drp1, while MIEFs are less selective and recruit both active and inactive Drp1 as well as oligomerization- or GTPase-deficient Drp1 mutants to mitochondria. Moreover, all the fission-incompetent Drp1 mutants tested (except the monomeric mutant K668E) affect Drp1-driven mitochondrial dynamics via incorporation of the mutants into the native oligomers to form function-deficient Drp1 assemblies. We here confirm that MIEFs also serve as a platform facilitating the binding of Drp1 to Mff and loss of MIEFs severely impairs the interaction between Drp1 and Mff. Collectively, our findings suggest that Mff and MIEFs respond differently to the molecular assembly state of Drp1 and that the extent of Drp1 oligomerization regulates mitochondrial dynamics.

Glucocorticoid receptor quaternary structure drives chromatin occupancy and transcriptional outcome.

Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.

OR26-6 Glucocorticoid Receptor Quaternary Structure Drives Chromatin Occupancy and Transcriptional Outcome

The multimeric structure adopted by transcription factor complexes at their genomic sites of interaction is a longstanding problem in transcription biology. Information bearing on this question is often inferred from indirect data sources. Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Glucocorticoid receptor (GR)’s dimeric/monomeric status is widely believed to drive pharmacological output of synthetic glucocorticoids. A widely discussed model suggests that dimeric GR regulates unfavorable metabolic pathways, while monomeric GR is responsible for anti-inflammatory activities. However, recent studies have indicated that dimeric GR is equally active in anti-inflammatory activities. Furthermore, quantitative fluorescence microscopy techniques in live cells show that the GR likely forms tetramers on enhancers during gene activation. These results suggest that higher oligomerization states are important for the gene regulatory responses of GR. To unveil the relationship between GR’s oligomerization and transcriptional outcome, we developed a GR null cell system to study how wild type GR and three different receptor oligomerization mutants bind chromatin and affect transcription at the genomic level. All mutants display unique quaternary structure characteristics; DNA-binding domain dimerization deficient (GRdim, A465T), monomeric (GRmon, A465T/I634A), and tetrameric (GRtetra, P481R) receptor. GRtetra mimics the DNA-bound conformation and forms tetramers independent of DNA binding. Integration of ChIP-seq, ATAC-seq, and RNA-seq data indicates that GR must form dimers to bind even pre-accessible chromatin, as monomeric GR is essentially a non-functional receptor. In comparison, GRtetra, acts as a super receptor by binding glucocorticoid response elements not accessible to the wild type receptor. This super receptor behavior can be observed in two distinct cell types. The sites occupied only by GRtetra in a given cell line are bound by the wild type GR in numerous other cell types, such as liver, macrophages, and pre-adipose cells. This suggest that the sites are authentic response elements. In addition, GRtetra both induces and, more importantly, represses more genes in response to hormone than the wild type GR. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher order structure that drives both the activating and repressive actions of glucocorticoids. This has important implications for the therapeutic uses of steroid hormones and the goal of finding selective anti-inflammatory drugs that do not create unwanted side-effects.

Crystal structure of dimeric Synechococcus spermidine synthase with bound polyamine substrate and product.

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

Quantifying the Oligomeric States of Membrane Proteins in Cells through Super-Resolution Localizations.

Transitions between different oligomeric states of membrane proteins are essential for proper cellular functions. However, the quantification of their oligomeric states in cells is technically challenging. Here we developed a new method to quantify oligomeric state(s) of highly expressed membrane proteins using the probability density function of molecule density ( PDFMD) calculated from super-resolution localizations. We provided the theoretical model of PDFMD, discussed the effects of protein concentration, cell geometry, and photophysics of fluorescent proteins on PDFMD, and provided experimental criteria for proper quantification of oligomeric states. This method was further validated using simulated single-molecule fluorescent movies and applied to two membrane proteins, UhpT and SbmA in E. coli. The study shows that PDFMD is useful in quantifying oligomeric states of membrane proteins in cells that can help in understanding cellular tasks. Potential applications to proteins with higher oligomeric states under high concentration and limitations of our methodology were also discussed.

Linker engineering in anti-TAG-72 antibody fragments optimizes biophysical properties, serum half-life, and high-specificity tumor imaging

Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scFVs, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L-purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G4S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G4S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G4S as a fast, accurate, and specific tumor-imaging agent.

Sensing Native Protein Solution Structures Using a Solid-state Nanopore: Unraveling the States of VEGF

Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely corresponding to monomer, dimer, and higher oligomeric states. Upon changing from a non-reducing to a reducing condition, ion-current blockage events from the monomeric state dominate, consistent with the expected reduction of the two inter-chain VEGF disulfide bonds. Cleavage by plasmin and application of either a positive or a negative NP bias results in nanopore signals corresponding either to the VEGF receptor recognition domain or to the heparin binding domain, accordingly. Interestingly, multi-level analysis of VEGF events reveals how individual domains affect their translocation pattern. Our study shows that careful characterization of ssNP results elucidates real-time structural information about the protein, thereby complementing classical techniques for structural analysis of proteins in solution with the added advantage of quantitative single-molecule resolution of native proteins.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Crystal structure of cbbF from Zymomonas mobilis and its functional implication

A phosphate group at the C1-atom of inositol-monophosphate (IMP) and fructose-1,6-bisphosphate (FBP) is hydrolyzed by a phosphatase IMPase and FBPase in a metal-dependent way, respectively. The two enzymes are almost indiscernible from each other because of their highly similar sequences and structures. Metal ions are bound to residues on the β1- and β2-strands and one mobile loop. However, FBP has another phosphate and FBPases exist as a higher oligomeric state, which may discriminate FBPases from IMPases. There are three genes annotated as FBPases in Zymomonas mobilis, termed also cbbF (ZmcbbF). The revealed crystal structure of one ZmcbbF shows a globular structure formed by five stacked layers. Twenty-five residues in the middle of the sequence form an α-helix and a β-strand, which occupy one side of the catalytic site. A non-polar Leu residue among them is protruded to the active site, pointing out unfavorable access of a bulky charged group to this side. In vitro assays have shown its dimeric form in solution. Interestingly, two β-strands of β1 and β2 are disordered in the ZmcbbF structure. These data indicate that ZmcbbF might structurally belong to IMPase, and imply that its active site would be reorganized in a yet unreported way.

Oligomerization Interface of RAGE Receptor Revealed by MS-Monitored Hydrogen Deuterium Exchange

Activation of the receptor for advanced glycation end products (RAGE) leads to a chronic proinflammatory signal, affecting patients with a variety of diseases. Potentially beneficial modification of RAGE activity requires understanding the signal transduction mechanism at the molecular level. The ligand binding domain is structurally uncoupled from the cytoplasmic domain, suggesting receptor oligomerization is a requirement for receptor activation. In this study, we used hydrogen-deuterium exchange and mass spectrometry to map structural differences between the monomeric and oligomeric forms of RAGE. Our results indicated the presence of a region shielded from exchange in the oligomeric form of RAGE and led to the identification of a new oligomerization interface localized at the linker region between domains C1 and C2. Based on this finding, a model of a RAGE dimer and higher oligomeric state was constructed.

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.

Modeling Complex between FBA and TIM: Functional Motions of FBA and TIM are Preserved in their Complex

Fructuose bisphostate aldolase (FBA) and triosephosphate isomerase (TIM) are the fourth and fifth enzymes in the glycolysis pathway and they are known to bind. FBA cleaves the six-carbon fructose 1, 6-bisphosphate into two three-carbon components – dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). TIM converts DHAP into GAP, a substrate for the subsequent synthetic step. These two alpha/beta barrel proteins have high structural similarity – with a core RMSD 4.8A. These two enzymes have low activity in the monomeric form with the functionally active structures present in higher oligomeric states. By applying Elastic Network Model, we find the modes of motions that are functionally important for these proteins. We build models for the complex between these two proteins to investigate their important motions in their complexes as well as for their different oligomeric states including those that are different in different species to learn their important modes of motions for different functionalities. For each protein, by multiple sequence alignment across the species, we predict the coevolving residues and cluster these residues along the structure. We build the information transfer pathways from the important interface residues to the catalytic residues. Change in these pathways in different oligomeric states maybe be related to the change of motions in the catalytic region in different oligomeric states. We use this knowledge about the changes in motions and the information transfer pathways within the structure of these interacting proteins as constraints for selecting the computational docked models of complexes between these two proteins to preserve their functional motions.

Mapping the VDAC1 Oligomerization Contact Sites and N-Terminus Translocation

Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. Accumulating evidence implicates the voltage-dependent anion channel (VDAC) as functioning in mitochondria-mediated apoptosis involving cytochrome c release, leading to caspases activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c conducting channel remain unknown. Previously, we demonstrated that apoptosis induction was accompanied by VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using Bioluminescence Resonance Energy Transfer technology. Moreover, apoptosis inhibitors inhibited VDAC oligomerization and a correlation between the levels of VDAC oligomerization and apoptosis was observed. Here, we combined site-directed mutagenesis with chemical cross-linking to reveal the contact sites between VDAC1 molecules in dimers and higher oligomers. Replacing hydrophobic amino acids with charged amino acids in b-strands 1,2 and 19, but not 14, interfered with VDAC1 oligomerization and apoptosis induction. Cysteine cross-linking results, from introducing cysteine at a defined position in cysteine-less VDAC1 and applying the cysteine-specific cross-linker, BMOE, supported the close vicinity of b-strands 1,2 and 19 in VDAC1 dimer. Moreover, the results suggest that VDAC1 exists as a dimer that undergoes conformational changes upon apoptosis induction to assemble into a higher oligomeric state. Additionally we demonstrated that the N-terminal region of VDAC1 lies inside the pore, but could also move and interact with the N-terminus from a second molecule to form a dimer. Our results suggest that the glycine rich sequence 21-GYGFG-25 is involved in the N-terminus translocation from the internal pore to the channel face. These results provide structural insight into cellular VDAC1's oligomeric state and its N-terminal region location and translocation.

Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment–protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (> 200 μmol photons m −2 s −1) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment–protein complexes function as low light-induced mobile antennae. Further, we have detected α-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.

Identification of Two Proteins Associated with Mammalian ATP Synthase

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.

Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states.

Fucoxanthin-chlorophyll proteins were purified from the centric diatom Cyclotella meneghiniana. Two major fractions were observed that differed in their polypeptide composition and oligomeric state. Trimers consist of mainly 18 kDa polypeptides. Higher oligomers are tightly assembled from different trimers, which contain mostly 19 kDa subunits. In both oligomeric states, the excitation energy coupling between fucoxanthin and chlorophyll a was preserved, and chlorophyll c was shown to transfer energy efficiently to chlorophyll a. Circular dichroism spectra showed close interaction between fucoxanthin and chlorophyll a, and different chlorophyll a molecules were demonstrated to interact excitonically. The assembly of trimers of antenna proteins with a distinct subunit composition into higher oligomeric states was not reported so far and differs from the situation found in higher plants. The differences in the supramolecular structure of the fucoxanthin-chlorophyll proteins reflect the dissimilar arrangement of the thylakoid membranes in diatoms, which lack the grana-stroma distinction.

Importance of low-oligomeric-weight species for prion propagation in the yeast prion system Sup35/Hsp104.

The [PSI+] determinant of Saccharomyces cerevisiae, consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers. The molecular chaperone Hsp104 is required to maintain self-replication of [PSI+]. We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup355-26, results in the dissociation of oligomeric Sup35 into monomeric species. Several intermediates of Sup355-26 could be detected during this process. Strong interactions are found between Hsp104 and hexameric/tetrameric Sup355-26, whereas the intermediate and monomeric "release" forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments. Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup355-26. No interaction can be detected between Hsp104 and higher oligomeric states (>/=8) of Sup355-26. Taking into account the fact that Hsp104 is required for maintenance of [PSI+], we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast.

Determination of native oligomeric state and substrate specificity of rat NTPDase1 and NTPDase2 after heterologous expression in Xenopus oocytes.

NTPDase1 and NTPDase2 are two related plasma membrane-located enzymes involved in the extracellular degradation of nucleoside 5'-tri- and -diphosphates. They differ regarding their hydrolysis ratios for ATP and ADP. Both enzymes have a predicted transmembrane domain close to the N- and C-terminus, respectively, connected by an extensive extracellular domain that carries the active site. We expressed the rat-derived enzymes in Xenopus laevis oocytes and analyzed their quarternary structure. As revealed by application of blue native PAGE and a comparison of glutaraldehyde cross-linking, native NTPDase1 and NTPDase2 occur in oligomeric form. Oligomer formation of the cell surface-located pool of the enzymes was verified by surface iodination. The two enzymes differed in oligomeric structure and in oligomer complex stability. NTPDase1 preferentially occurred as a dimer that could be dissociated into monomeric forms in the presence of Coomassie Brilliant blue G-250 and dithiothreitol whereas NTPDase2 revealed higher oligomeric forms up to tetramers, largely resistant to dithiothreitol. Our results further suggest that the enzymes exist in varying oligomeric states. In contrast to NTPDase1, substrate specificity of NTPDase2 was altered with prolonged expression time, resulting in a decrease in the ATPase/ADPase activity ratio from 10 : 1 to 2.5 : 1. This was accompanied by a transition into a higher oligomeric state. Our results suggest that despite close sequence identity, NTPDase1 and NTPDase2 differ in oligomeric structure. Dynamic alterations in oligomeric state may induce changes in substrate preference and thus influence the pattern of extracellular nucleotide degradation in situ.

A mutation in the receiver domain of the Agrobacterium tumefaciens transcriptional regulator VirG increases its affinity for operator DNA

We fused the wild-type Agrobacterium tumefaciens virG gene and the constitutive virGN54D allele to the malE gene of Escherichia coli, and studied the binding of MBP-VirG fusions to the autoregulated virG promoter. MBP-VirGN54D protein bound this promoter with 10-fold higher affinity than MBP-VirG, and bound to vir box I with eightfold higher affinity than to vir box III. Disruption of vir box III did not alter the affinity for vir box I, suggesting a lack of cooperativity between these sites. We provide evidence that protein bound at a single vir box may have a higher oligomeric state than non-bound protein, and that a DNA distortion adjacent to vir box I may occur during activation.