Stabilization of adalimumab Fab through the introduction of disulfide bonds between the variable and constant domains.

Fab is a promising format for antibody drug. Therefore, efforts have been made to improve its thermal stability for therapeutic and commercial use. So far, we have attempted to introduce a disulfide bond into the Fab fragment to improve its thermal stability and demonstrated that it is possible to do this without sacrificing its biochemical function. In this study, to develop a novel stabilization strategy for Fab, we attempted to introduce a disulfide bond between the variable and constant domains and prepared three variants of Fab; H:G10C + H:P210C, L:P40C + L:E165C, and H:G10C + H:P210C + L:P40C + L:E165C. Differential scanning calorimetry measurements showed that each of these variants had improved thermal stability. In addition, the variants with two disulfide bonds demonstrated a 6.5 °C increase in their denaturation temperatures compared to wild-type Fab. The introduction of disulfide bonds was confirmed by X-ray crystallography, and the variants retained their antigen-binding activity. The variants were also found to be less aggregative than the wild type. Our results demonstrate that the introduction of a disulfide bond between the variable and constant domains significantly improves the thermal stability of Fab.

Function and Structure of a Terpene Synthase Encoded in a Giant Virus Genome.

Giant viruses are nonstandard viruses with large particles and genomes. While previous studies have shown that their genomes contain various sequences of interest, their genes related specifically to natural product biosynthesis remain unexplored. Here we analyze the function and structure of a terpene synthase encoded by the gene of a giant virus. The enzyme is phylogenetically separated from the terpene synthases of cellular organisms; however, heterologous gene expression revealed that it still functions as a terpene synthase and produces a cyclic terpene from a farnesyl diphosphate precursor. Crystallographic analysis revealed its protein structure, which is relatively compact but retains essential motifs of the terpene synthases. We thus suggest that like cellular organisms, giant viruses produce and utilize natural products for their ecological strategies.

Experimental phasing opportunities for macromolecular crystallography at very long wavelengths.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

Crystal Structure Analysis of SH2 Domains in Complex with Phosphotyrosine Peptides.

While the number of tertiary structures solved by cryoelectron microscopy has rapidly increased, X-ray crystallography is still a popular method to determine the tertiary structure of proteins at atomic resolution. However, there are still problems associated with X-ray crystallography, including crystallization and crystal twinning. Indeed, we encountered crystallization and twinning problems in the crystal structure analysis of the SH2 domains complexed with a phosphorylated peptide derived from the oncoprotein CagA. In this chapter, we describe the methods used to overcome these problems. In addition, we provide details of the optimization of the crystallization conditions and cryo-conditions, which are usually not given in published crystal structure analyses.

Functional molecular evolution of a GTP sensing kinase: PI5P4Kß.

Over 4 billion years of evolution, multiple mutations, including nucleotide substitutions, gene and genome duplications and recombination, have established de novo genes that translate into proteins with novel properties essential for high-order cellular functions. However, molecular processes through which a protein evolutionarily acquires a novel function are mostly speculative. Recently, we have provided evidence for a potential evolutionary mechanism underlying how, in mammalian cells, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) evolved into a GTP sensor from ATP-utilizing kinase. Mechanistically, PI5P4Kß has acquired the guanine efficient association (GEA) motif by mutating its nucleotide base recognition sequence, enabling the evolutionary transition from an ATP-dependent kinase to a distinct GTP/ATP dual kinase with its KM for GTP falling into physiological GTP concentrations-the genesis of GTP sensing activity. Importantly, the GTP sensing activity of PI5P4Kß is critical for the manifestation of cellular metabolism and tumourigenic activity in the multicellular organism. The combination of structural, biochemical and biophysical analyses used in our study provides a novel framework for analysing how a protein can evolutionarily acquire a novel activity, which potentially introduces a critical function to the cell.

A unique peptide-based pharmacophore identifies an inhibitory compound against the A-subunit of Shiga toxin.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can cause fatal systemic complications. Recently, we identified a potent inhibitory peptide that binds to the catalytic A-subunit of Stx. Here, using biochemical structural analysis and X-ray crystallography, we determined a minimal essential peptide motif that occupies the catalytic cavity and is required for binding to the A-subunit of Stx2a, a highly virulent Stx subtype. Molecular dynamics simulations also identified the same motif and allowed determination of a unique pharmacophore for A-subunit binding. Notably, a series of synthetic peptides containing the motif efficiently inhibit Stx2a. In addition, pharmacophore screening and subsequent docking simulations ultimately identified nine Stx2a-interacting molecules out of a chemical compound database consisting of over 7,400,000 molecules. Critically, one of these molecules markedly inhibits Stx2a both in vitro and in vivo, clearly demonstrating the significance of the pharmacophore for identifying therapeutic agents against EHEC infection.

Ligand Recognition by the Lipid Transfer Domain of Human OSBP Is Important for Enterovirus Replication.

Oxysterol-binding protein (OSBP), which transports cholesterol and phosphatidylinositol 4-monophosphate (PtdIns[4]P) between different organelles, serves as a conserved host factor for the replication of various viruses, and OSBP inhibitors exhibit antiviral effects. Here, we determined the crystal structure of the lipid transfer domain of human OSBP in complex with endogenous cholesterol. The hydrocarbon tail and tetracyclic ring of cholesterol interact with the hydrophobic tunnel of OSBP, and the hydroxyl group of cholesterol forms a hydrogen bond network at the bottom of the tunnel. Systematic mutagenesis of the ligand-binding region revealed that M446W and L590W substitutions confer functional tolerance to an OSBP inhibitor, T-00127-HEV2. Employing the M446W variant as a functional replacement for the endogenous OSBP in the presence of T-00127-HEV2, we have identified previously unappreciated amino acid residues required for viral replication. The combined use of the inhibitor and the OSBP variant will be useful in elucidating the enigmatic in vivo functions of OSBP.

The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

Exploration and structure-based engineering of alkenal double bond reductases catalyzing the CαCß double bond reduction of coniferaldehyde.

Lignin, a complex aromatic polymer, represents a significant obstacle in lignocellulosic biomass utilization. The polymerization of lignin occurs by radical couplings, which mainly form ether and C-C bonds between monolignol units. The chemical stability of these bonds between monolignol units causes the recalcitrant nature of lignin. Since the Cα-Cß double bond in the monolignols is a crucial chemical feature for the radical coupling, reduction of the double bond would decrease the degree of lignin polymerization, avoiding the recalcitrance of lignin. To develop a method of lignin engineering, we have focused on alkenal double bond reductases (DBR), which can reduce the Cα-Cß double bond of a monolignol precursor. Here, a novel bacterial DBR from Parvibaculum lavamentivorans DS-1 (PlDBR) was found. This enzyme can reduce the side-chain double bond of coniferaldehyde (CALD) and has a 41% amino-acid sequence identity with CALD DBR from Arabidopsis thaliana (AtDBR). The crystal structure of the PlDBR showed that it has a larger substrate-binding pocket than AtDBR, conferring broader substrate specificity on the former. Structural and mutation analyses of PlDBR and AtDBR suggested that Tyr51 and Try252 are critical residues for the catalytic activity of PlDBR. In addition, Tyr81 of AtDBR appears to cause substrate inhibition. Replacing Tyr81 of AtDBR with a smaller amino-acid residue, as in the AtDBR variants Tyr81Leu and Tyr81Ala, resulted in a substantially higher CALD-reducing activity compared to the wild type. These variants would be promising candidates for lignin manipulation to decrease the recalcitrance of lignocellulosic biomass.

Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti.

BACKGROUND: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated. RESULTS: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein. CONCLUSION: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels.

3CL Protease Inhibitors with an Electrophilic Arylketone Moiety as Anti-SARS-CoV-2 Agents.

The novel coronavirus, SARS-CoV-2, has been identified as the causative agent for the current coronavirus disease (COVID-19) pandemic. 3CL protease (3CLpro) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CLpro. The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CLpro. Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.

C-Glycoside metabolism in the gut and in nature: Identification, characterization, structural analyses and distribution of C-C bond-cleaving enzymes.

C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.

FAD-dependent C-glycoside-metabolizing enzymes in microorganisms: Screening, characterization, and crystal structure analysis.

C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme "C-glycoside 3-oxidase" named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.

Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin.

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx.

Characterization of a novel type of carbonic anhydrase that acts without metal cofactors.

BACKGROUND: Carbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and bicarbonate (HCO3-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, ß, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution. RESULTS: Here, we report that a previously uncharacterized protein group, named "COG4337," constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO2 hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+ß barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO2 metabolism in these organelles. CONCLUSIONS: COG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.

Crystal structure of the full-length LysR-type transcription regulator CbnR in complex with promoter DNA.

LysR-type transcription regulators (LTTRs) comprise one of the largest families of transcriptional regulators in bacteria. They are typically homo-tetrameric proteins and interact with promoter DNA of ~ 50-60 bp. Earlier biochemical studies have suggested that LTTR binding to promoter DNA bends the DNA and, upon inducer binding, the bend angle of the DNA is reduced through a quaternary structure change of the tetrameric LTTR, leading to the activation of transcription. To date, crystal structures of full-length LTTRs, DNA-binding domains (DBD) with their target DNAs, and the regulatory domains with and without inducer molecules have been reported. However, these crystal structures have not provided direct evidence of the quaternary structure changes of LTTRs or of the molecular mechanism underlying these changes. Here, we report the first crystal structure of a full-length LTTR, CbnR, in complex with its promoter DNA. The crystal structure showed that, in the absence of bound inducer molecules, the four DBDs of the tetrameric CbnR interact with the promoter DNA, bending the DNA by ~ 70°. Structural comparison between the DNA-free and DNA-bound forms demonstrates that the quaternary structure change of the tetrameric CbnR required for promoter region-binding arises from relative orientation changes of the three domains in each subunit. The mechanism of the quaternary structure change caused by inducer binding is also discussed based on the present crystal structure, affinity analysis between CbnR and the promoter DNA, and earlier mutational studies on CbnR. DATABASE: Atomic coordinates and structure factors for the full-length Cupriavidus necator NH9 CbnR in complex with promoter DNA are available in the Protein Data Bank under the accession code 7D98.

Correction to: Structural basis for a highly (S)-enantioselective reductase towards aliphatic ketones with only one carbon difference between side chain.

The original version of this article contains error for some of the authors corrections were not included during correction stage.

Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction.

Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.

Structural basis for a highly (S)-enantioselective reductase towards aliphatic ketones with only one carbon difference between side chain.

Aliphatic ketones, such as 2-butanone and 3-hexanone, with only one carbon difference among side chains adjacent to the carbonyl carbon are difficult to be reduced enantioselectively. In this study, we utilized an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD) to reduce challenging aliphatic ketones such as 2-butanone (methyl ethyl ketone) and 3-hexanone (ethyl propyl ketone) to their corresponding (S)-alcohols with 94% ee and > 99% ee, respectively. Through crystallographic structure determination, it was suggested that residue Trp288 limit the size of the small binding pocket. Docking simulations imply that Trp288 plays an important role to form a C-H⋯π interaction for proper orientation of ketones in the pro-S binding pose in order to produce (S)-alcohols. The excellent (S)-enantioselectivity is due to a non-productive pro-R binding pose, consistent with the observation that the (R)-alcohol acts as an inhibitor of (S)-alcohol oxidation.

Structural and Computational Bases for Dramatic Skeletal Rearrangement in Anditomin Biosynthesis.

AndA, an Fe(II)/α-ketoglutarate (αKG)-dependent enzyme, is the key enzyme that constructs the unique and congested bridged-ring system of anditomin (1), by catalyzing consecutive dehydrogenation and isomerization reactions. Although we previously characterized AndA to some extent, the means by which the enzyme facilitates this drastic structural reconstruction have remained elusive. In this study, we have solved three X-ray crystal structures of AndA, in its apo form and in the complexes with Fe(II), αKG, and two substrates. The crystal structures and mutational experiments identified several key amino acid residues important for the catalysis and provided insight into how AndA controls the reaction. Furthermore, computational calculations validated the proposed reaction mechanism for the bridged-ring formation and also revealed the requirement of a series of conformational changes during the transformation.