Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Cryo-EM structures of Trypanosoma brucei gambiense ISG65 with human complement C3 and C3b and their roles in alternative pathway restriction.

African Trypanosomes have developed elaborate mechanisms to escape the adaptive immune response, but little is known about complement evasion particularly at the early stage of infection. Here we show that ISG65 of the human-infective parasite Trypanosoma brucei gambiense is a receptor for human complement factor C3 and its activation fragments and that it takes over a role in selective inhibition of the alternative pathway C5 convertase and thus abrogation of the terminal pathway. No deposition of C4b, as part of the classical and lectin pathway convertases, was detected on trypanosomes. We present the cryo-electron microscopy (EM) structures of native C3 and C3b in complex with ISG65 which reveal a set of modes of complement interaction. Based on these findings, we propose a model for receptor-ligand interactions as they occur at the plasma membrane of blood-stage trypanosomes and may facilitate innate immune escape of the parasite.

A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein.

Identification of a protein minimal fragment amenable to crystallisation can be time- and labour intensive especially if large amounts are required and the protein has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design of a minimal expression construct. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellular, eukaryotic proteins. Eukaryotic expression systems provide an alternative but are costly, slow and require special handling and equipment. Using a member of a structurally uncharacterized, eukaryotic receptor family as an example we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) guided construct design in conjunction with truncation scanning and targeted expression host switching to identify a minimal expression construct that can be produced with high yields and moderate costs.

Estrogen suppresses HOXB2 expression via ERα in breast cancer cells.

The expression of HOXB2, a homeobox transcription factor, is altered in a variety of solid tumors. Using an in vivo screen to identify regulators of breast tumor growth in murine mammary fat pads, Boimel and co-workers recently identified HOXB2 as a tumor suppressor. However, the mechanistic underpinnings of its role in breast cancer is not understood. Given the emerging interaction of estrogen-regulated gene expression and altered HOX gene expression network in the pathophysiology of breast cancer, this study addressed the relationship between estrogen signaling and HOXB2 expression. Using a mouse model and human breast cancer cell lines, we show that estrogen suppresses HOXB2 expression. Suppression of HOXB2 by PPT, a known ERα agonist, in MCF-7 and T47D cells indicated the involvement of ERα, which was confirmed by siRNA-mediated ERα knockdown experiments. In-silico analysis of the upstream promoter region revealed the presence of three putative EREs. Chromatin immunoprecipitation experiments showed that upon estrogen binding, ERα engaged with EREs in the 5' upstream region of HOXB2 in MCF-7 and T47D cells. Future investigations should address the implications of estrogen-mediated suppression on the proposed tumor suppressor function of HOXB2.

Molecular Basis for Bordetella pertussis Interference with Complement, Coagulation, Fibrinolytic, and Contact Activation Systems: the Cryo-EM Structure of the Vag8-C1 Inhibitor Complex.

Complement, contact activation, coagulation, and fibrinolysis are serum protein cascades that need strict regulation to maintain human health. Serum glycoprotein, a C1 inhibitor (C1-INH), is a key regulator (inhibitor) of serine proteases of all the above-mentioned pathways. Recently, an autotransporter protein, virulence-associated gene 8 (Vag8), produced by the whooping cough pathogen, Bordetella pertussis, was shown to bind to C1-INH and interfere with its function. Here, we present the structure of the Vag8-C1-INH complex determined using cryo-electron microscopy at a 3.6-Å resolution. The structure shows a unique mechanism of C1-INH inhibition not employed by other pathogens, where Vag8 sequesters the reactive center loop of C1-INH, preventing its interaction with the target proteases.IMPORTANCE The structure of a 10-kDa protein complex is one of the smallest to be determined using cryo-electron microscopy at high resolution. The structure reveals that C1-INH is sequestered in an inactivated state by burial of the reactive center loop in Vag8. By so doing, the bacterium is able to simultaneously perturb the many pathways regulated by C1-INH. Virulence mechanisms such as the one described here assume more importance given the emerging evidence about dysregulation of contact activation, coagulation, and fibrinolysis leading to COVID-19 pneumonia.

Human RAD51 paralogue RAD51C fosters repair of alkylated DNA by interacting with the ALKBH3 demethylase.

The integrity of our DNA is challenged daily by a variety of chemicals that cause DNA base alkylation. DNA alkylation repair is an essential cellular defence mechanism to prevent the cytotoxicity or mutagenesis from DNA alkylating chemicals. Human oxidative demethylase ALKBH3 is a central component of alkylation repair, especially from single-stranded DNA. However, the molecular mechanism of ALKBH3-mediated damage recognition and repair is less understood. We report that ALKBH3 has a direct protein-protein interaction with human RAD51 paralogue RAD51C. We also provide evidence that RAD51C-ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adduct located within 3'-tailed DNA, which serves as a substrate for the RAD51 recombinase. We further show that the lack of RAD51C-ALKBH3 interaction affects ALKBH3 function in vitro and in vivo. Our data provide a molecular mechanism underlying upstream events of alkyl adduct recognition and repair by ALKBH3.

Development of bi-functional chimeric enzyme (CtGH1-L1-CtGH5-F194A) from endoglucanase (CtGH5) mutant F194A and ß-1,4-glucosidase (CtGH1) from Clostridium thermocellum with enhanced activity and structural integrity.

Site-directed mutagenesis of ß-1,4-endoglucanase from family 5 glycoside hydrolase (CtGH5) from Clostridium thermocellum was performed to develop a mutant CtGH5-F194A that gave 40 U/mg specific activity against carboxymethyl cellulose, resulting 2-fold higher activity than wild-type CtGH5. CtGH5-F194A was fused with a ß-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. The chimera (CtGH1-L1-CtGH5-F194A) expressed as a soluble protein using E. coli BL-21cells displaying 3- to 5-fold higher catalytic efficiency for endoglucanase and ß-glucosidase activities. TLC analysis of hydrolysed product of CMC by chimera 1 revealed glucose as final product confirming both ß-1,4-endoglucanase and ß-1,4-glucosidase activities, while the products of CtGH5-F194A were cellobiose and cello-oligosaccharides. Protein melting studies of CtGH5-F194A showed melting temperature (Tm), 68 °C and of CtGH1, 79 °C, whereas, chimera showed 78 °C. The improved structural integrity, thermostability and enhanced bi-functional enzyme activities of chimera makes it potentially useful for industrial application in converting biomass to glucose and thus bioethanol.

The multi-ligand binding first family 35 Carbohydrate Binding Module (CBM35) of Clostridium thermocellum targets rhamnogalacturonan I.

Carbohydrate Binding Modules (CBMs) targeting cellulose, xylan and mannan have been reported, however, a CBM targeting rhamnogalacturonan I (RG I) has never been identified. We had studied earlier a rhamnogalacturonan lyase (CtRGL) from Clostridium thermocellum that was associated with a family 35 CBM, Rgl-CBM35. In this study we show that Rgl-CBM35 displays binding with ß-d-glucuronic acid (ß-D-GlcpA), Δ4,5-anhydro-d-galactopyranosyluronic acid (Δ4,5-GalpA), rhamnogalacturonan I, arabinan, galactan, glucuronoxylans and arabinoxylans. Rgl-CBM35 contains a conserved ligand binding site in the loops known for binding ß-D-GlcpA and Δ4,5-GalpA moiety of unsaturated RG I and pectic-oligosaccharides. Mutagenesis revealed that Asn118 plays an important role in binding ß-D-GlcpA, Δ4,5-GalpA, sugarbeet arabinan and potato galactan at its conserved ligand binding site present in surface exposed loops. EDTA-treated Rgl-CBM35 showed no affinity towards ß-D-GlcpA and Δ4,5-GalpA underscoring Ca2+ mediated ligand recognition. Contrastingly, the EDTA-treated Rgl-CBM35 and its mutant N118A displayed affinity for sugarbeet arabinan and potato galactan. The curtailed affinity of Y37A/N118A and R69A/N118A double mutants towards sugarbeet arabinan emphasized the presence of a second ligand binding site. Rgl-CBM35 is the first CBM reported to primarily target RG I and also is the first member of family 35 CBM possessing at least two ligand binding sites.

Novel insights into the degradation of ß-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase.

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a ß-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ±â€¯0.12 mg/ml and Vmax, 109 ±â€¯1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of ß-1,3-glucan hydrolysed products released initially, showed ß-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Šresolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 ß-strands forming a four-domain structure viz. a ß-sandwich domain I at N-terminal, an α/ß-domain II, an (α/α)6 barrel domain III, and a small 5-stranded ß-sandwich domain IV.

SAXS and homology modelling based structure characterization of pectin methylesterase a family 8 carbohydrate esterase from Clostridium thermocellum ATCC 27405.

Pectin methylesterase (CtPME) from Clostridium thermocellum of family 8 carbohydrate esterase (CE8) belongs to pectin methylesterase super family (E.C.3.1.1.11). BLAST analysis of CtPME showed 38% sequence identity with PME from Erwinia chrysanthemi. Multiple sequence alignment of CtPME with other known structures of pectin methylesterase revealed the conserved and semi-conserved amino acid residues. Homology modelling of CtPME structure revealed a characteristic right handed parallel ß-helices. The energy of modelled structure was minimized by using YASARA software. The Ramachandran plot of CtPME shows 83.7% residues in non-glycine and non-proline residues in most-favorable region, 13.8% in additional allowed region and 1.4% in generously allowed region, indicating that CtPME has a stable conformation. The secondary structure of CtPME predicted using PSI-Pred software and confirmed by the circular dichroism (CD) showed α-helices (3.1%), ß-sheets (40.1%) and random coils (56.9%). Small Angle X-ray Scattering (SAXS) analysis demonstrated the overall shape and structural characterization of CtPME in solution form. Guinier analysis gave the radius of gyration (Rg) 2.28 nm for globular shape and 0.74 nm for rod shape. Kratky plot gave the indication that protein is fully folded in solution. The ab initio derived dummy atom model of CtPME superposed well on modelled CtPME structure.

Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans.

The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α)6 incomplete toroidal fold and a layered ß sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% ß sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×105) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during ß-elimination.

Insights into Structure and Reaction Mechanism of β-Mannanases.

ß-mannanases have been shown to play an important role in various biological processes such as the cell wall component degradation, defence signalling in plants, the mobilization of storage reserves and in various industrial processes. To date, glycoside hydrolases (GHs) have been divided into 135 families and 14 clans from A to N based upon their sequence, overall structural fold and function. ß -mannanases belong glycoside hydrolases and exist under four different glycoside hydrolase families, GH5, GH26, GH113 and GH134. GH5 and GH26 are combined in clan GH-A. GH5 and GH26 contain hydrolases which follow the retaining type reaction mechanism. Structural survey of ß- mannanases of GH5 and GH26, suggests that both families contain similar TIM barrel fold. In addition, they also share common catalytic residues and their location in the structure. Despite these structural similarities, a distinct difference lies between the substrate binding sub-sites which define substrate specificity. This review summarizes the recent reports on the structure and function perspectives of ß- mannanases of GH5 and GH26 and highlights the similarities and differences between them.

A New Member of Family 11 Polysaccharide Lyase, Rhamnogalacturonan Lyase (CtRGLf) from Clostridium thermocellum.

A thermostable, alkaline rhamnogalacturonan lyase (RG lyase) CtRGLf, of family 11 polysaccharide lyase from Clostridium thermocellum was cloned, expressed, purified and biochemically characterised. Both, the full-length CtRGLf (80 kDa) protein and its truncated derivative CtRGL (63.9 kDa) were expressed as soluble proteins and displayed maximum activity against rhamnogalacturonan I (RG I). CtRGLf showed maximum activity at 70 °C, while CtRGL at 60 °C. Both enzymes showed maximum activity at pH 8.5. CtRGLf and CtRGL do not show higher activity on substrates with high ß-D-galactopyranose (D-Galp) substitution, this catalytic property deviates from that of some earlier characterised RG lyases which prefer substrates with high D-Galp substitution. The enzyme activity of CtRGLf and CtRGL was enhanced by 1.5 and 1.3 fold, respectively, in the presence of 3 mM of Ca(2+) ions. The TLC analysis of the degraded products of RG I, released by the action of CtRGLf and CtRGL revealed the production of RG oligosaccharides as major products confirming their endolytic activity.