Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes.

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

The solution structure of the heavy chain-only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions.

Heavy chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy in vivo. Here, we have characterized the solution arrangement of the molecule. Two 1443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

Structure of the DNA repair helicase XPD.

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

Non-pharmaceutical interventions for COVID-19: a systematic review on environmental control measures.

The purpose of this review was to identify the effectiveness of environmental control (EC) non-pharmaceutical interventions (NPIs) in reducing transmission of SARS-CoV-2 through conducting a systematic review. EC NPIs considered in this review are room ventilation, air filtration/cleaning, room occupancy, surface disinfection, barrier devices, [Formula: see text] monitoring and one-way-systems. Systematic searches of databases from Web of Science, Medline, EMBASE, preprint servers MedRxiv and BioRxiv were conducted in order to identify studies reported between 1 January 2020 and 1 December 2022. All articles reporting on the effectiveness of ventilation, air filtration/cleaning, room occupancy, surface disinfection, barrier devices, [Formula: see text] monitoring and one-way systems in reducing transmission of SARS-CoV-2 were retrieved and screened. In total, 13 971 articles were identified for screening. The initial title and abstract screening identified 1328 articles for full text review. Overall, 19 references provided evidence for the effectiveness of NPIs: 12 reported on ventilation, 4 on air cleaning devices, 5 on surface disinfection, 6 on room occupancy and 1 on screens/barriers. No studies were found that considered the effectiveness of [Formula: see text] monitoring or the implementation of one-way systems. Many of these studies were assessed to have critical risk of bias in at least one domain, largely due to confounding factors that could have affected the measured outcomes. As a result, there is low confidence in the findings. Evidence suggests that EC NPIs of ventilation, air cleaning devices and reduction in room-occupancy may have a role in reducing transmission in certain settings. However, the evidence was usually of low or very low quality and certainty, and hence the level of confidence ascribed to this conclusion is low. Based on the evidence found, it was not possible to draw any specific conclusions regarding the effectiveness of surface disinfection and the use of barrier devices. From these results, we further conclude that community agreed standards for well-designed epidemiological studies with low risk of bias are needed. Implementation of such standards would enable more confident assessment in the future of the effectiveness of EC NPIs in reducing transmission of SARS-CoV-2 and other pathogens in real-world settings. This article is part of the theme issue 'The effectiveness of non-pharmaceutical interventions on the COVID-19 pandemic: the evidence'.

Structure-function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways.

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes-C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC-are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.

Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.

The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.

Next generation Glucose-1-phosphate thymidylyltransferase (RmlA) inhibitors: An extended SAR study to direct future design.

The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by glucose-1-phosphate thymidylyltransferase (RmlA), which condenses glucose-1-phosphate (Glu-1-P) with deoxythymidine triphosphate (dTTP) to yield dTDP-d-glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor's pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.

Periplasmic depolymerase provides insight into ABC transporter-dependent secretion of bacterial capsular polysaccharides.

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogen Salmonella enterica serovar Typhi produces "Vi antigen" CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of the Burkholderiales, which are paradoxically distinguished from S. Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel ß-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced into S Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.

Engineering of a Peptide α-N-Methyltransferase to Methylate Non-Proteinogenic Amino Acids.

Introduction of α-N-methylated non-proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre-methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α-N-automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C-terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non-proteinogenic amino acids has not been addressed. An engineered OphMA, intein-mediated protein ligation and solid-phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non-natural amides. This approach may have application in the biotechnological production of therapeutic peptides.

Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity.

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.

The Biosynthesis of the Benzoxazole in Nataxazole Proceeds via an Unstable Ester and has Synthetic Utility.

Heterocycles, a class of molecules that includes oxazoles, constitute one of the most common building blocks in current pharmaceuticals and are common in medicinally important natural products. The antitumor natural product nataxazole is a model for a large class of benzoxazole-containing molecules that are made by a pathway that is not characterized. We report structural, biochemical, and chemical evidence that benzoxazole biosynthesis proceeds through an ester generated by an ATP-dependent adenylating enzyme. The ester rearranges via a tetrahedral hemiorthoamide to yield an amide, which is a shunt product and not, as previously thought, an intermediate in the pathway. A second zinc-dependent enzyme catalyzes the formation of hemiorthoamide from the ester but, by shuttling protons, the enzyme eliminates water, a reverse hydrolysis reaction, to yield the benzoxazole and avoids the amide. These insights have allowed us to harness the pathway to synthesize a series of novel halogenated benzoxazoles.

Insights into the Mechanism of the Cyanobactin Heterocyclase Enzyme.

Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.

Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase.

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters.

Biotin is an essential vitamin in plants and mammals, functioning as the carbon dioxide carrier within central lipid metabolism. Bacterial pimeloyl-CoA synthetase (BioW) acts as a highly specific substrate-selection gate, ensuring the integrity of the carbon chain in biotin synthesis. BioW catalyzes the condensation of pimelic acid (C7 dicarboxylic acid) with CoASH in an ATP-dependent manner to form pimeloyl-CoA, the first dedicated biotin building block. Multiple structures of Bacillus subtilis BioW together capture all three substrates, as well as the intermediate pimeloyl-adenylate and product pyrophosphate (PPi), indicating that the enzyme uses an internal ruler to select the correct dicarboxylic acid substrate. Both the catalytic mechanism and the surprising stability of the adenylate intermediate were rationalized through site-directed mutagenesis. Building on this understanding, BioW was engineered to synthesize high-value heptanoyl (C7) and octanoyl (C8) monocarboxylic acid-CoA and C8 dicarboxylic-CoA products, highlighting the enzyme's synthetic potential.

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function.

With advances in sequencing technology, uncharacterized proteins and domains of unknown function (DUFs) are rapidly accumulating in sequence databases and offer an opportunity to discover new protein chemistry and reaction mechanisms. The focus of this review, the formerly enigmatic YcaO superfamily (DUF181), has been found to catalyze a unique phosphorylation of a ribosomal peptide backbone amide upon attack by different nucleophiles. Established nucleophiles are the side chains of Cys, Ser, and Thr which gives rise to azoline/azole biosynthesis in ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products. However, much remains unknown about the potential for YcaO proteins to collaborate with other nucleophiles. Recent work suggests potential in forming thioamides, macroamidines, and possibly additional post-translational modifications. This review covers all knowledge through mid-2016 regarding the biosynthetic gene clusters (BGCs), natural products, functions, mechanisms, and applications of YcaO proteins and outlines likely future research directions for this protein superfamily.

Complexes formed by the siderophore-based monosulfactam antibiotic BAL30072 and their interaction with the outer membrane receptor PiuA of P. aeruginosa.

Nuclear magnetic resonance and infrared spectroscopy have been used to investigate the formation of complexes of BAL30072 with Fe3+ and Ga3+ in solution and to collect geometrical parameters supporting reliable 3D structure models. Structural models for the ligand-metal complexes with different stoichiometries have been characterized using density functional theory calculations. Blind ensemble docking to the PiuA receptor from P. aeruginosa was performed for the different complexes to compare binding affinities and statistics of the residues most frequently contacted. When compared to analogues, BAL30072 was found to have an intrinsic propensity to form complexes with low ligand-to-metal stoichiometry. By using one of the sulfate oxygen atoms as a third donor in addition to the bidentate pyridinone moiety, BAL30072 can form a L2M complex, which was predicted to be the one with the best binding affinity to PiuA. The example of BAL30072 strongly suggests that a lower stoichiometry might be the one recognized by the receptor, so that to focus only on the highest stoichiometry might be misleading for siderophores with less than six donors.

Oxidation of the Cyanobactin Precursor Peptide Is Independent of the Leader Peptide and Operates in a Defined Order.

The five-membered nitrogen plus heteroatom rings known as azolines or in their oxidized form as azoles are very common in natural products and drugs. The oxidation of thiazoline to thiazole in the cyanobactin class of natural products is one of the several important transformations that are known to alter the biological properties of the compound. The ordering of the various chemical reactions that occur during cyanobactin biosynthesis is not fully understood. The structure of the flavin-dependent enzyme responsible for the oxidation of multiple thiazolines reveals it contains an additional domain that in other enzymes recognizes linear peptides. We characterize the activity of the enzyme on two substrates: one with a peptide leader and one without. Kinetics and biophysics reveal that the leader on the substrate is not recognized by the enzyme. The enzyme is faster on either substrate than the macrocyclase or protease in vitro. The enzyme has a preferred order of oxidation of multiple thiazolines in the same linear peptide.

TonB-Dependent Receptor Repertoire of Pseudomonas aeruginosa for Uptake of Siderophore-Drug Conjugates.

The conjugation of siderophores to antimicrobial molecules is an attractive strategy to overcome the low outer membrane permeability of Gram-negative bacteria. In this Trojan horse approach, the transport of drug conjugates is redirected via TonB-dependent receptors (TBDR), which are involved in the uptake of essential nutrients, including iron. Previous reports have demonstrated the involvement of the TBDRs PiuA and PirA from Pseudomonas aeruginosa and their orthologues in Acinetobacter baumannii in the uptake of siderophore-beta-lactam drug conjugates. By in silico screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The piuD gene in LESB58 is located at the same genetic locus as piuA in strain PAO1. PiuD has a similar crystal structure as PiuA and is involved in the transport of the siderophore-drug conjugates BAL30072, MC-1, and cefiderocol in strain LESB58. To screen for additional siderophore-drug uptake systems, we overexpressed 28 of the 34 TBDRs of strain PAO1 and identified PfuA, OptE, OptJ, and the pyochelin receptor FptA as novel TBDRs conferring increased susceptibility to siderophore-drug conjugates. The existence of a TBDR repertoire in P. aeruginosa able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.

Synthesis of the natural product descurainolide and cyclic peptides from lignin-derived aromatics.

Alternative sources of potential feedstock chemicals are of increasing importance as the availability of oil decreases. The biopolymer lignin is viewed as a source of useful mono-aromatic compounds as exemplified by the industrial scale production of vanillin from this biomass. Alternative lignin-derived aromatics are available in pure form but to date examples of the use of these types of compounds are rare. Here we address this issue by reporting the conversion of an aromatic keto-alcohol to the anti- and syn-isomers of Descurainolide A. The key step involves a rhodium-catalyzed allylic substitution reaction. Enantio-enriched allylic alcohols were generated via an isothiourea-catalyzed kinetic resolution enabling access to both the (2R,3R) and (2S,3S) enantiomers of anti-Descurainolide A. In addition we show that the lignin-derived keto-alcohols can be converted into unnatural amino acid derivatives of tyrosine. Finally, these amino acids were incorporated into cyclic peptide scaffolds through the use of both chemical and an enzyme-mediated macrocylisation.