Hexameric Structure Research Articles

Human mitochondrial AAA+ ATPase SKD3/CLPB assembles into nucleotide-stabilized dodecamers

SKD3, also known as human CLPB, belongs to the AAA+ family of ATPases associated with various activities. Mutations in the SKD3/CLPB gene cause 3-methylglutaconic aciduria type VII and congenital neutropenia. SKD3 is upregulated in acute myeloid leukemia, where it contributes to anti-cancer drug resistance. SKD3 resides in the mitochondrial intermembrane space, where it forms ATP-dependent high-molecular weight complexes, but its biological function and mechanistic links to the clinical phenotypes are currently unknown. Using sedimentation equilibrium and dynamic light scattering, we show that SKD3 is monomeric at low protein concentration in the absence of nucleotides, but it forms oligomers at higher protein concentration or in the presence of adenine nucleotides. The apparent molecular weight of the nucleotide-bound SKD3 is consistent with self-association of 12 monomers. Image-class analysis and averaging from negative-stain electron microscopy (EM) of SKD3 in the ATP-bound state visualized cylinder-shaped particles with an open central channel along the cylinder axis. The dimensions of the EM-visualized particle suggest that the SKD3 dodecamer is formed by association of two hexameric rings. While hexameric structure has been often observed among AAA+ ATPases, a double-hexamer sandwich found for SKD3 appears uncommon within this protein family. A functional significance of the non-canonical structure of SKD3 remains to be determined.

In silico investigation of symptom development model based on coat protein interactions of two cucumber mosaic virus strains

Cucumber mosaic virus (CMV) causes serious diseases in agricultural crops worldwide, predominantly in vegetable and ornamental plants. A critical interaction has been reported between the plant Ferredoxin I (Fd I) protein with the coat protein (CP) of the M strain of CMV (M-CMV) but not by the CP of the Q strain of CMV (Q-CMV). To ascertain the most likely symptom development model based on CMV CP interacting with the plant Fd I protein, further in silico investigation was performed on the CPs of the strains Q-CMV and M-CMV. The biological assembly of the CMV CP in virions is composed of hexameric and pentameric units. The results showed that there were significantly different interactions between pentameric and hexameric forms of M and Q-CMV CPs with Fd I. Pentameric structural forms of M-CMV CP interact strongly with Fd I compared to pentameric structures of Q-CMV CP, while there were no significant differences in the affinities of hexameric structures for Fd I between M and Q-CMV CPs. it can be deduced that probably pentameric structures of M-CMV CP block activation of plant Fd I protein. Therefore, it could be hypothesized that trapping Fd I in the complex of pentameric structures can lead to inhibition of the activity of plant Fd I in the photosynthetic electron transport chain leading the severe symptom development seen in M-CMV infected plants but not in Q-CMV infected plants. It seems likely that any factor that inhibits the interaction of CMV CPs with plant Fd I can prevent this symptom development.

Bacterial antagonism of Chromobacterium haemolyticum and characterization of its putative type VI secretion system

This study reports the isolation of a new Chromobacterium haemolyticum strain named WI5 from a hydroponic farming facility. WI5 exhibited remarkable bacterial antagonistic properties, eliminating Salmonella, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus (initial inoculum load ∼105 CFU/ml) in dual-species co-culture biofilms. Antagonism was strictly contact-dependent and highly influenced by nutrient availability. Next, we identified a complete suite of putative Type VI secretion system (T6SS) genes in the WI5 genome, annotated the gene locus architecture, and determined the crystal structure of hallmark T6SS tube protein Hcp1, which revealed a hexameric ring structure with an outer and inner diameter of 77 and 45 Å, respectively. Structural comparison with homologs showed differences in the key loops connecting the β−strands in which the conserved residues are located, suggesting a role of these residues in the protein function. The T6SS is well-known to facilitate interbacterial competition, and the putative T6SS characterized herein might be responsible for the remarkable antagonism by C. haemolyticum WI5. Collectively, these findings shed light on the nature of bacterial antagonism and a putative key virulence determinant of C. haemolyticum, which might aid in further understanding its potential ecological role in natural habitats.

Structural basis of DegP protease temperature-dependent activation.

Protein quality control is an essential cellular function mainly executed by a vast array of different proteases and molecular chaperones. One of the bacterial high temperature requirement A (HtrA) protein family members, the homo-oligomeric DegP protease, plays a crucial role in the Escherichia coli protein quality control machinery by removing unfolded proteins or preventing their aggregation and chaperoning them to their final folded state within the periplasm. DegP contains two regulatory PDZ domains, which play key roles in substrate recognition and in the transformation of DegP between inactive hexameric and proteolytic active cage-like structures. Here, we analyze the interaction and dynamics of the DegP PDZ domains underlying this transformation by high-resolution NMR spectroscopy complemented with biochemical cleavage assays. We identify an interdomain molecular lock, which controls the interactions between the two PDZ domains, regulated by fine-tuned temperature-dependent protein dynamics, and which is potentially conserved in proteins harboring tandem PDZ domains.

Structural Stability of Insulin Oligomers and Protein Association-Dissociation Processes: Free Energy Landscape and Universal Role of Water.

Association and dissociation of proteins are important biochemical events. In this Feature Article, we analyze the available studies of these processes for insulin oligomers in aqueous solution. We focus on the solvation of the insulin monomer in water, stability and dissociation of its dimer, and structural integrity of the hexamer. The intricate role of water in solvation of the dimer- and hexamer-forming surfaces, in long-range interactions between the monomers and the stability of the oligomers, is discussed. Ten water molecules inside the central cavity stabilize the structure of the insulin hexamer. We discuss how different order parameters can be used to understand the dissociation of the insulin dimer. The calculation of the rate using a recently computed multidimensional free energy provides considerable insight into the interplay between protein and water dynamics.

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

Three-megadalton complex of methanogenic electron-bifurcating and CO2-fixing enzymes.

The first reaction of the methanogenic pathway from carbon dioxide (CO2) is the reduction and condensation of CO2 to formyl-methanofuran, catalyzed by formyl-methanofuran dehydrogenase (Fmd). Strongly reducing electrons for this reaction are generated by heterodisulfide reductase (Hdr) in complex with hydrogenase or formate dehydrogenase (Fdh) using a flavin-based electron-bifurcation mechanism. Here, we report enzymological and structural characterizations of Fdh-Hdr-Fmd complexes from Methanospirillum hungatei. The complexes catalyze this reaction using electrons from formate and the reduced form of the electron carrier F420. Conformational changes in HdrA mediate electron bifurcation, and polyferredoxin FmdF directly transfers electrons to the CO2 reduction site, as evidenced by methanofuran-dependent flavin-based electron bifurcation even without free ferredoxin, a diffusible electron carrier between Hdr and Fmd. Conservation of Hdr and Fmd structures suggests that this complex is common among hydrogenotrophic methanogens.

Structural Analysis of an Octameric Resorcinarene Self-Assembly in Toluene and its Morphological Transition by Temperature.

Calix[4]resorcinarene derivatives such as C-undecylcalix[4]resorcinarene self-assemble into a hexameric capsule-like structure. This structure is expected in any apolar solvent, but we recently found a self-assembled octameric form of resorcinarene in toluene. To understand this unexpected form, we performed small-angle X-ray scattering (SAXS) measurements of the octameric self-assembly. In ab initio shape reconstruction and model fitting of the SAXS profile, a core-shell spherical structure resembling a reverse micelle was found in the octameric self-assembly. The shell and core were composed of alkyl chains and resorcinol moieties, respectively. We also evaluated the temperature dependence of the octameric self-assembly. Above 95 °C, the structure began directly decomposing into unimers, implying the partial cleavage of hydrogen bonds in the octamer core. Meanwhile, below 25 °C, the spherical structure of the octamer partially transformed to a cylindrical structure. This morphological transition could be understood by packing parameter theory, which is often applied to suspected micellar structures.

CTLA4-FasL, the Hexameric Targeted FasL Fusion Protein, As Potential Novel Treatment for DLBCL

The bi-functional drug CTLA4-FasL (KAHR-102) is a fusion protein composed of the extracellular domains of CTLA4 and FasL. It naturally forms a stable homo-hexamer capable of targeting two different cellular receptors; the CTLA4 domains target B7 receptors (CD80 and CD86) on cells bearing these receptors, while the FasL domains target functional Fas-receptors (CD95) to induce apoptosis. Although CTLA4-FasL can induce apoptosis in all cells expressing functional Fas receptors, it is at least 100 times more potent on cells expressing both B7 and functional Fas receptors. The higher efficacy in cells expressing both receptors is adjoined by robust activation of pro-apoptotic intracellular pathways in conjunction to abrogation of anti-apoptotic ones. This unique combined activity cannot be detected in cells bearing just Fas receptors and can be blocked by preventing the binding of the protein to CD80 and CD86. Both B7 and functional Fas receptors are expressed on lymphoma cells, mainly of the B cell lineage, making them exceptionally sensitive to the CTLA4-FasL apoptotic effect.CTLA4-FasL was found to be highly efficient in inducing apoptosis in different resistant DLBCL cell-lines bearing translocations such as , FARAGE, OCI-LY7, OCI-LY19 and SUD-HL-4. Now we show that CTLA4-FasL specifically binds to CD80 and CD86 on these cells, hinders their viability with EC50 of 0.02-0.06 nM (MTS assay), and induces robust apoptosis in these cells (annexin V-PI assay). CTLA4-FasL's potency is correlated with CD80, CD86 and CD95 expression on the cell's surface.Importantly, CTLA4-FasL significantly improves mouse survival in a systemic lymphoma model induced by IV injection of FARAGE DLBCL cells into the tail vein of irradiated NUDE mice (4 SC injections, every other day). When given at a dose of 10mg/mice/injection its effect was comparable to that of 350 mg/mice of Rituximab.Previously, we evaluated CTLA4-FasL's toxicity in GLP studies in mice and cynomolgus monkeys. No gross pathology or adverse clinical symptoms were observed in mice (n=76) or monkeys (n=10) treated with the highest dose of CTLA4-FasL (0.5 mg/kg in mice, 0.125 mg/kg in monkeys). Transient and dose-dependent leukopenia was observed in monkeys only and this was resolves in 7 days. As leukopenia was observed in monkeys only and was not evident in mice treated with the same adjusted dose, we now compared Fas receptor level of expression and CTLA4-FasL 's binding to mice, monkeys and human PBMCs, and also CTLA4-FasL 's ability to induce apoptosis of the different PBMCs. Interestingly, the monkeys' cells express more Fas receptors and bind the protein better than both human and mice cells. In accordance with that, significantly more apoptosis of monkeys' PBMCs was evident when cells were treated with CTLA4-FasL.Though leukopenia was not observed in mice, it is still possible that some population of cells are affected by CTLA4-FasL. In order to test CTLA4-FasL's effect on different immune system cells in immune competent mice, BALB/C mice were treated with CTLA4-FasL and 24h later peripheral blood and spleens were harvested and cells were immuno-stained and analyzed by flow-cytometry. A significant decrease in percentage and actual counts of B and T lymphocytes (CD4 and CD8) as well as macrophages and monocytes was importantly accompanied by an increase in percentage and actual cell number of neutrophils and NK cells.In summary, the fusion protein CTLA4-FasL, with its unique hexameric structure and bi-functional mode of targeted action, was shown to be an effective treatment for human-mouse xenograft model of DLBCL. Transient and dose-dependent leukopenia was the only significant adverse effect, seen only in monkeys, and is maybe explained by the higher sensitivity of monkeys' cells to the protein's apoptotic effect. It might also be the result of the longer half-life of the protein in monkeys when compared to mice. The sparing and even increase in the number of neutrophils and NK cells might indicate that the innate immune system will be preserved after treatment with CTLA4-FasL. DisclosuresAronin:KAHR Medical: Employment. Ben Gigi-Tamir:KAHR Medical: Employment. Amsili:KAHR Medical: Employment, Other: Options. Sagiv:KAHR Medical: Employment. Gozlan:KAHR mEDICAL: Employment. Shani:KAHR Medical: Employment, Other: OPTIONS, Patents & Royalties. Dranitzki Elhalel:KAHR Medical: Consultancy, Other: Options, Patents & Royalties: PATENTS, Research Funding.

Putative hexameric glycosyltransferase functional unit revealed by the crystal structure of Acinetobacter baumannii MurG.

Lipid II, the main component of the bacterial cell wall, is synthesized by the addition of UDP-N-acetylglucosamine to the UDP-N-acetylmuramic acid pentapeptide catalyzed by the glycosyltransferase MurG. Owing to its critical role in cell-wall biosynthesis, MurG is considered to be an attractive target for antibacterial agents. Although the Mur family ligases have been extensively studied, the molecular mechanism of the oligomeric scaffolding assembly of MurG remains unclear. In this study, MurG from Acinetobacter baumannii (abMurG), a human pathogen, was characterized and its hexameric crystal structure was unveiled; this is the first homo-oligomeric structure to be described in the MurG family and the Mur family. Homogeneous protein samples were produced for structural studies using size-exclusion chromatography, the absolute molecular mass was calculated via multi-angle light scattering, and protein-protein interactions were analyzed using the PDBePISA server. abMurG was found to form homo-oligomeric complexes in solution, which might serve as functional units for the scaffolding activity of MurG. Furthermore, analysis of this structure revealed the molecular assembly mechanism of MurG. This structural and biochemical study elucidated the homo-oligomerization mechanism of MurG and suggests a new potential antibiotic target on MurG.

Supercoiling Structure-Based Design of a Trimeric Coiled-Coil Peptide with High Potency against HIV-1 and Human β-Coronavirus Infection.

Hexameric structure formation through packing of three C-terminal helices and an N-terminal trimeric coiled-coil core has been proposed as a general mechanism of class I enveloped virus entry. In this process, the C-terminal helical repeat (HR2) region of viral membrane fusion proteins becomes transiently exposed and accessible to N-terminal helical repeat (HR1) trimer-based fusion inhibitors. Herein, we describe a mimetic of the HIV-1 gp41 HR1 trimer, N3G, as a promising therapeutic against HIV-1 infection. Surprisingly, we found that in addition to protection against HIV-1 infection, N3G was also highly effective in inhibiting infection of human β-coronaviruses, including MERS-CoV, HCoV-OC43, and SARS-CoV-2, possibly by binding the HR2 region in the spike protein of β-coronaviruses to block their hexameric structure formation. These studies demonstrate the potential utility of anti-HIV-1 HR1 peptides in inhibiting human β-coronavirus infection. Moreover, this strategy could be extended to the design of broad-spectrum antivirals based on the supercoiling structure of peptides.

Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10.

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rβ, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rβ binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

Split conformation of Chaetomium thermophilum Hsp104 disaggregase

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

Functional recombinant human complement C1q with different affinity tags

Complement C1q is a multifunctional protein able to sense pathogens and immune molecules such as immunoglobulins and pentraxins, and to trigger the classical complement pathway through activation of its two associated proteases, C1r and C1s. C1q is a multimeric protein composed of three homologous yet distinct polypeptide chains A, B, and C, each composed of an N-terminal collagen-like sequence and a C-terminal globular gC1q module, that assemble into six heterotrimeric (A-B-C) subunits. This hexameric structure exhibits the characteristic shape of a bouquet of flowers, comprising six collagen-like triple helices, each terminating in a trimeric C-terminal globular head. We have produced previously functional recombinant full-length C1q in stably transfected HEK 293-F cells, with a FLAG tag inserted at the C-terminal end of C1qC chain. We report here the generation of additional recombinant C1q proteins, with a FLAG tag fused to the C-terminus of C1qA or C1qB chains, or to the N-terminus of the C1qC chain. Two other variants harboring a Myc or a 6-His tag at the C-terminal end of C1qC were also produced. We show that all C1q variants, except for the His-tagged protein, can be produced at comparable yields and are able to bind with similar affinities to either IgM, a ligand of the globular regions, or to the C1r2-C1s2 tetramer, and to trigger IgM-mediated serum complement activation. These new recombinant C1q variants provide additional tools to investigate the multiple functions of C1q.

Rings and Chains: Synthesis and Characterization of Polyferrocenylmethylene.

The synthesis and characterization of polyferrocenylmethylene (PFM) starting from dilithium 2,2-bis(cyclopentadienide)propane and a Me2 C[1]magnesocenophane is reported. Molecular weights of up to Mw = 11700g mol-1 featuring a dispersity, Ð, of 1.40 can be achieved. The material is studied by different methods comprising nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry, differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) measurements elucidating the molecular structure and thermal properties of these novel polymers. Moreover, cyclic voltammetry (CV) reveals quasi-reversible oxidation and reduction behavior and communication between the iron centers. Also, the crystal structure of a related cyclic hexamer is presented.

The Unexpected Base‐Pairing Behavior of Cyanuric Acid in RNA and Ribose versus Cyanuric Acid Induced Helicene Assembly of Nucleic Acids: Implications for the Pre‐RNA Paradigm

Cyanuric acid heterocycle (CA) forms supramolecular structures with adenine nucleobases/nucleosides and oligonucleotides leading to speculation that they can act as forerunners to RNA. Herein, we studied the assembly behavior of RNA containing CA and CA-ribose nucleoside. Contrary to previous reports, CA in RNA, and as the ribonucleoside, leads to destabilization of supramolecular assemblies, which led to reevaluation of the CA-adenine hexameric rosette structure. We propose an unprecedented non-covalent supramolecular helicene structure to account for the striking difference in behavior, which has implications for novel paradigms for reorganizing the structures of nucleic acids, synthesis of long helicenes, and pre-RNA world paradigms. The results caution against extrapolating the self-assembly behavior of individual heterocycles from the level of monomers to oligomers - since the base-paring properties of (non)canonical nucleobases are impacted by the type of oligomeric backbone to which they are attached.

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

Resolving the Mechanisms of Soy Glycinin Self-Coacervation and Hollow-Condensate Formation.

Self-coacervation of animal-derived proteins has been extensively investigated while that of plant proteins remains largely unexplored. Here, we study the process of soy glycinin self-coacervation and transformation into hollow condensates. The protein hexameric structure composed of hydrophilic and hydrophobic polypeptides is crucial for coacervation. The process is driven by charge screening of the intrinsically disordered region of acidic polypeptides, allowing for weak hydrophobic interactions between exposed hydrophobic polypeptides. We find that the coacervate surface exhibits order, which stabilizes the coacervate shape during hollow-condensate formation. The latter process occurs via nucleation and growth of protein-poor phase in the coacervate interior, during which another ordered layer at the inner surface is formed. Aging enhances the stability of both coacervates and hollow condensates. Understanding plant protein coacervation holds promises for fabricating novel functional materials.

The comparative analysis of the properties and structures of purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27

Two recombinant purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27 encoded by genes TT_C1070 (TthPNPI) and TT_C0194 (TthPNPII) were purified and characterized. The comparative analysis of their sequences, molecular weight, enzymes specificity and kinetics of the catalyzed reaction were realized. As a result, it was determined that the TthPNPI is specific to guanosine while the TthPNPII to adenosine. According to the results of the size exclusion chromatography and SAXS study both enzymes are hexameric molecules. Based on the sequence alignment with homologous purine nucleoside phosphorylases (PNPs), Asn was identified as a purine base recognizing residue in the active site of TthPNPI and Asp in TthPNPII. The three-dimensional structure of TthPNPII was solved at 2.5 Å resolution by molecular replacement method using crystals grown in microgravity. Position of phosphate in the active site cavity is located. The possible arrangement of adenosine and guanosine in TthPNPII active site cavity is considered using superposition with the structures of homologous trimeric and hexameric PNPs complexed with corresponding substrates. The peculiarities of oligomeric structure of TthPNPII in comparison with homologous PNPs are described. It is shown that two trimeric molecules of TthPNPII in the asymmetric part of the unit cell are connected by three two-fold axis into a hexamer with 32-point symmetry. This type of hexameric structure of PNP is found for the first time. The interface area between the subunits in trimeric molecule and between the trimers in TthPNPII hexamer is described. Communicated by Ramaswamy H. Sarma.