Holliday junction resolution by At-HIGLE: an SLX1 lineage endonuclease from Arabidopsis thaliana with a novel in-built regulatory mechanism.

Holliday junction is the key homologous recombination intermediate, resolved by structure-selective endonucleases (SSEs). SLX1 is the most promiscuous SSE of the GIY-YIG nuclease superfamily. In fungi and animals, SLX1 nuclease activity relies on a non-enzymatic partner, SLX4, but no SLX1-SLX4 like complex has ever been characterized in plants. Plants exhibit specialized DNA repair and recombination machinery. Based on sequence similarity with the GIY-YIG nuclease domain of SLX1 proteins from fungi and animals, At-HIGLE was identified to be a possible SLX1 like nuclease from plants. Here, we elucidated the crystal structure of the At-HIGLE nuclease domain from Arabidopsis thaliana, establishing it as a member of the SLX1-lineage of the GIY-YIG superfamily with structural changes in DNA interacting regions. We show that At-HIGLE can process branched-DNA molecules without an SLX4 like protein. Unlike fungal SLX1, At-HIGLE exists as a catalytically active homodimer capable of generating two coordinated nicks during HJ resolution. Truncating the extended C-terminal region of At-HIGLE increases its catalytic activity, changes the nicking pattern, and monomerizes At-HIGLE. Overall, we elucidated the first structure of a plant SLX1-lineage protein, showed its HJ resolving activity independent of any regulatory protein, and identified an in-built novel regulatory mechanism engaging its C-terminal region.

A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein.

BACKGROUND: The influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation. METHODS: The phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging. RESULTS: The four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells. CONCLUSION: This structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.

Microscopic and Transcriptomic Analyses of Dalbergoid Legume Peanut Reveal a Divergent Evolution Leading to Nod-Factor-Dependent Epidermal Crack-Entry and Terminal Bacteroid Differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Recognition and processing of branched DNA substrates by Slx1-Slx4 nuclease.

Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3' end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures.

Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.

Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine. These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase. Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.

Structural evaluation of a mimicry-recognizing paratope: plasticity in antigen-antibody interactions manifests in molecular mimicry.

Molecular mimicry manifests antagonistically with respect to the specificity of immune recognition. However, it often occurs because different Ags share surface topologies in terms of shape or chemical nature. It also occurs when a flexible paratope accommodates dissimilar Ags by adjusting structural features according to the antigenic epitopes or differential positioning in the Ag combining site. Toward deciphering the structural basis of molecular mimicry, mAb 2D10 was isolated from a maturing immune response elicited against methyl α-d-mannopyranoside and also bound equivalently to a dodecapeptide. The physicochemical evidence of this carbohydrate-peptide mimicry in the case of mAb 2D10 had been established earlier. These studies had strongly suggested direct involvement of a flexible paratope in the observed mimicry. Surprisingly, comparison of the Ag-free structure of single-chain variable fragment 2D10 with those bound to sugar and peptide Ags revealed a conformationally invariant state of the Ab while binding to chemically and structurally disparate Ags. This equivalent binding of the two dissimilar Ags was through mutually independent interactions, demonstrating functional equivalence in the absence of structural correlation. Thus, existence of a multispecific, mature Ab in the secondary immune response was evident, as was the plasticity in the interactions while accommodating topologically diverse Ags. Although our data highlight the structural basis of receptor multispecificity, they also illustrate mechanisms adopted by the immune system to neutralize the escape mutants generated during pathogenic insult.

Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase via Raman crystallography.

Y-Family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. (13)C- and (15)N-labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg(2+) and Mn(2+), nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of *C modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that was possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn(2+) or Mg(2+), the primer extension reaction did not occur; instead, a ternary protein·template·d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features caused by ring modes from the template/primer bases being perturbed and from the DNA backbone appear, as well as features from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary complex than in the binary complex. Using standardized Raman intensities followed as a function of time, the C(*C)TP population in the crystal was maximal at ∼20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred.

Cavity architecture based modulation of ligand binding tunnels in plant START domains.

The Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain represents an evolutionarily conserved superfamily of lipid transfer proteins widely distributed across the tree of life. Despite significant expansion in plants, knowledge about this domain remains inadequate in plants. In this work, we explore the role of cavity architectural modulations in START protein evolution and functional diversity. We use deep-learning approaches to generate plant START domain models, followed by surface accessibility studies and a comprehensive structural investigation of the rice START family. We validate 28 rice START domain models, delineate binding cavities, measure pocket volumes, and compare these with mammalian counterparts to understand evolution of binding preferences. Overall, plant START domains retain the ancestral α/ß helix-grip signature, but we find subtle variation in cavity architectures, resulting in significantly smaller ligand-binding tunnels in the plant kingdom. We identify cavity lining residues (CLRs) responsible for reduction in ancestral tunnel space, and these appear to be class specific, and unique to plants, providing a mechanism for the observed shift in domain function. For instance, mammalian cavity lining residues A135, G181 and A192 have evolved to larger CLRs across the plant kingdom, contributing to smaller sizes, minimal STARTs being the largest, while members of type-IV HD-Zip family show almost complete obliteration of lipid binding cavities, consistent with their present-day DNA binding functions. In summary, this work quantifies plant START structural & functional divergence, bridging current knowledge gaps.

Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion.

The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.

Crystal structure and functional insights of hemopexin fold protein from grass pea.

A regulatory protein from grass pea (Lathyrus sativus), LS-24, a close homolog of albumin 2 from garden pea (Pisum sativum) that is associated with polyamine biosynthesis, was characterized and the structure of a hemopexin-type fold among plant proteins illustrated. Crystal structure of LS-24 determined at 2.2 A resolution by multiple isomorphous replacement phasing showed four-bladed beta-propeller structure having a pseudo 4-fold molecular symmetry along a metal ion-binding central channel. The structure represents typical mammalian hemopexin fold with discernible features correlated with the possible functional variations. The protein was found to exist in the dimeric state. While LS-24 dimer binds to spermine in the crystal structure as well as in solution, binding of heme in solution resulted in the dissociation of the dimer into monomers with concomitant release of bound spermine. Interactions of heme and spermine with LS-24 bear physiological implications. While binding of spermine to LS-24 can be linked with polyamine biosynthesis that of heme correlates with oxidative stress. Mutually exclusive binding of heme and spermine in different oligomeric states suggest a role for LS-24 in sensing oxidative stress through a ligand-regulated monomer-dimer transition switch.

The structure of a haemopexin-fold protein from cow pea (Vigna unguiculata) suggests functional diversity of haemopexins in plants.

The haemopexin fold is present in almost all life forms and is utilized for carrying out diverse physiological functions. The structure of CP4, a haemopexin-fold protein from cow pea (Vigna unguiculata), was determined at 2.1 Šresolution. The protein exists as a monomer both in solution and in the crystal. The structure revealed a typical four-bladed ß-propeller topology. The protein exhibits 42% sequence similarity to LS-24 from Lathyrus sativus, with substantial differences in the surface-charge distribution and in the oligomeric state. A structure-based sequence analysis of haemopexin-fold proteins of plant and mammalian origin established a sequence signature associated with the haemopexin motif. This signature sequence enabled the identification of other proteins with possible haemopexin-like topology of both plant and animal origin. Although CP4 shares a structural fold with LS-24 and other haemopexins, biochemical studies indicated possible functional differences between CP4 and LS-24. While both of these proteins exhibit spermine-binding potential, CP4 does not bind to haem, unlike LS-24.

Structural Aspects of DNA Repair and Recombination in Crop Improvement.

The adverse effects of global climate change combined with an exponentially increasing human population have put substantial constraints on agriculture, accelerating efforts towards ensuring food security for a sustainable future. Conventional plant breeding and modern technologies have led to the creation of plants with better traits and higher productivity. Most crop improvement approaches (conventional breeding, genome modification, and gene editing) primarily rely on DNA repair and recombination (DRR). Studying plant DRR can provide insights into designing new strategies or improvising the present techniques for crop improvement. Even though plants have evolved specialized DRR mechanisms compared to other eukaryotes, most of our insights about plant-DRRs remain rooted in studies conducted in animals. DRR mechanisms in plants include direct repair, nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), non-homologous end joining (NHEJ) and homologous recombination (HR). Although each DRR pathway acts on specific DNA damage, there is crosstalk between these. Considering the importance of DRR pathways as a tool in crop improvement, this review focuses on a general description of each DRR pathway, emphasizing on the structural aspects of key DRR proteins. The review highlights the gaps in our understanding and the importance of studying plant DRR in the context of crop improvement.

A combined structural and biochemical approach reveals translocation and stalling of UvrB on the DNA lesion as a mechanism of damage verification in bacterial nucleotide excision repair.

Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules clamps a different DNA strand using its ß-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway.

Purification, identification and preliminary crystallographic studies of a 2S albumin seed protein from Lens culinaris.

Lens culinaris (lentil) is a widely consumed high-protein-content leguminous crop. A 2S albumin protein (26.5 kDa) has been identified using NH(2)-terminal sequencing from a 90% ammonium sulfate saturation fraction of total L. culinaris seed protein extract. The NH(2)-terminal sequence shows very high homology to PA2, an allergy-related protein from Pisum sativum. The 2S albumin protein was purified using a combination of size-exclusion and ion-exchange chromatography. Crystals of the 2S seed albumin obtained using the hanging-drop vapour-diffusion method diffracted to 2.5 A resolution and were indexed in space group P4(1) (or P4(3)), with unit-cell parameters a = b = 78.6, c = 135.2 A.

Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis.

Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P4(1) (or P4(3)), with unit-cell parameters a = b = 150.7, c = 164.9 A.

Structure and mechanism of nucleases regulated by SLX4.

SLX4 is a multidomain platform that regulates various proteins that are involved in genome maintenance and stability. Among these proteins are three structure-selective nucleases (SSEs). XPF-ERCC1 and MUS81-EME1 are structurally similar and function as heterodimers of highly similar subunits, in which only one is active. Two independent modules are formed from subunits of the heterodimers - a dimer of nuclease and nuclease-like domains and a dimer of tandem helix-hairpin-helix HhH2 domains. Both modules are responsible for substrate recognition. The third SSE, SLX1, contains GIY-YIG and RING domains and is a promiscuous nuclease. Structural data imply that SLX1 exists in free form as an autoinhibited homodimer. Association with SLX4 platform disrupts the homodimer and activates SLX1. This review discusses the available structural and mechanistic information on SLX4-regulated SSEs.

Structural and Mechanistic Analysis of the Slx1-Slx4 Endonuclease.

The SLX1-SLX4 endonuclease required for homologous recombination and DNA repair in eukaryotic cells cleaves a variety of branched DNA structures. The nuclease subunit SLX1 is activated by association with a scaffolding protein SLX4. At the present time, little is known about the structure of SLX1-SLX4 or its mechanism of action. Here, we report the structural insights into SLX1-SLX4 by detailing the crystal structure of Candida glabrata (Cg) Slx1 alone and in combination with the C-terminal region of Slx4. The structure of Slx1 reveals a compact arrangement of the GIY-YIG nuclease and RING domains, which is reinforced by a long α helix. Slx1 forms a stable homodimer that blocks its active site. Slx1-Slx4 interaction is mutually exclusive with Slx1 homodimerization, suggesting a mechanism for Slx1 activation by Slx4.