Structural Elucidation and Antiviral Activity of Covalent Cathepsin L Inhibitors.

Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC50 range in Vero E6 cells and inhibit CatL in the picomolar Ki range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile.

Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically important bacterium with such a system is Clostridioides difficile (formerly Clostridium). The CDTa and CDTb proteins from its system represent important therapeutic targets. CDTb has been proposed to be a di-heptamer, but its physiological heptameric structure has not yet been reported. Here, we report the cryo-EM structure of CDTa bound to the CDTb-pore, which reveals that CDTa binding induces partial unfolding and tilting of the first CDTa α-helix. In the CDTb-pore, an NSS-loop exists in 'in' and 'out' conformations, suggesting its involvement in substrate translocation. Finally, 3D variability analysis revealed CDTa movements from a folded to an unfolded state. These dynamic structural information provide insights into drug design against hypervirulent C. difficile strains.

Preparation of Clostridium perfringens binary iota-toxin pore complex for structural analysis using cryo-EM.

Iota toxin, a type of A-B toxin produced by Clostridium perfringens, comprises an enzymatic component (Ia) and a membrane-binding component (Ib). The translocation of Ia to the target cell via the pore formed by Ib allows it to function as an ADP-ribosyltransferase that inhibits actin polymerization in the host cell. The structure of Ia-bound Ib-pore has been determined using cryo-electron microscopy (cryo-EM), thereby elucidating the mechanism of the initial Ia translocation; however, open questions regarding Ia translocation still exist. In this chapter, we describe a new method of preparing Ia-bound Ib-pore complex samples for structural analysis at high resolution using cryo-EM. This method is different from previously reported methods for other A-B toxins. Consequently, it produces Ib-pore with two different states with short and long membrane-spanning ß-barrel stem. We expect that this method will be useful in functional and structural studies of iota toxin and other binary toxins.

Common Mechanism for Target Specificity of Protein- and DNA-Targeting ADP-Ribosyltransferases.

Many bacterial pathogens utilize ADP-ribosyltransferases (ARTs) as virulence factors. The critical aspect of ARTs is their target specificity. Each individual ART modifies a specific residue of its substrates, which could be proteins, DNA, or antibiotics. However, the mechanism underlying this specificity is poorly understood. Here, we review the substrate recognition mechanism and target residue specificity based on the available complex structures of ARTs and their substrates. We show that there are common mechanisms of target residue specificity among protein- and DNA-targeting ARTs.

A myelin sheath protein forming its lattice.

The formation of a mature, multilayered myelin sheath requires the compaction of lipid bilayers, but the molecular mechanism by which these bilayers condense is an open question. In this issue, Ruskamo et al. find that peripheral myelin protein P2 forms an ordered three-dimensional lattice within model membranes using Escherichia coli polar lipid liposomes. These data will help to understand the assembly, function, and structure of the myelin sheath.

Cryo-EM structures reveal translocational unfolding in the clostridial binary iota toxin complex.

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.

Substrate N2 atom recognition mechanism in pierisin family DNA-targeting, guanine-specific ADP-ribosyltransferase ScARP.

ScARP from the bacterium Streptomyces coelicolor belongs to the pierisin family of DNA-targeting ADP-ribosyltransferases (ARTs). These enzymes ADP-ribosylate the N2 amino groups of guanine residues in DNA to yield N2-(ADP-ribos-1-yl)-2'-deoxyguanosine. Although the structures of pierisin-1 and Scabin were revealed recently, the substrate recognition mechanisms remain poorly understood because of the lack of a substrate-binding structure. Here, we report the apo structure of ScARP and of ScARP bound to NADH and its GDP substrate at 1.50 and 1.57 Å resolutions, respectively. The bound structure revealed that the guanine of GDP is trapped between N-ribose of NADH and Trp-159. Interestingly, N2 and N3 of guanine formed hydrogen bonds with the OE1 and NE2 atoms of Gln-162, respectively. We directly observed that the ADP-ribosylating toxin turn-turn (ARTT)-loop, including Trp-159 and Gln-162, plays a key role in the specificity of DNA-targeting, guanine-specific ARTs as well as protein-targeting ARTs such as the C3 exoenzyme. We propose that the ARTT-loop recognition is a common substrate-recognition mechanism in the pierisin family. Furthermore, this complex structure sheds light on similarities and differences among two subclasses that are distinguished by conserved structural motifs: H-Y-E in the ARTD subfamily and R-S-E in the ARTC subfamily. The spatial arrangements of the electrophile and nucleophile were the same, providing the first evidence for a common reaction mechanism in these ARTs. ARTC (including ScARP) uses the ARTT-loop for substrate recognition, whereas ARTD (represented by Arr) uses the C-terminal helix instead of the ARTT-loop. These observations could help inform efforts to improve ART inhibitors.

Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity.

Aeromonas sobria serine protease (ASP) is an extracellular serine protease secreted by the organism. Here, we identified the amino acid residue of ASP that contributes to substrate specificity by using both synthetic peptides and biological protein components. The results showed that the arginine residue at position 566 (Arg-566) of ASP, which is located in the extra occluding region of ASP close to an entrance of the catalytic cavity, is involved in the substrate specificity. A substitutional point mutation of the Arg-566 residue of ASP to Ala residue (ASP[R566A]) caused a decrease of the proteolytic efficiency for a certain substrate. In addition, ASP lost the ability to recognize the primary substrate by such a point mutation, and ASP[R566A] reacted to a wide range of synthetic substrates. It is likely that Arg-566 causes an interaction with the amino acid residue at position P3 of the substrate, which is the third amino acid residue upstream from the cleavage site. Another study using ORF2 protein, a chaperone protein of ASP, further suggested that Arg-566 could also play an important role in interaction with ORF2. We therefore conclude that the Arg-566 residue of ASP is likely responsible for the selection of substrates.

Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.

Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia-actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and ß/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and ß/γ-actin, but its sensitivity towards ß/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates ß/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and ß/γ-actin.

Comparative Studies of Actin- and Rho-Specific ADP-Ribosylating Toxins: Insight from Structural Biology.

Mono-ADP-ribosylation is a major post-translational modification performed by bacterial toxins, which transfer an ADP-ribose moiety to a substrate acceptor residue. Actin- and Rho-specific ADP-ribosylating toxins (ARTs) are typical ARTs known to have very similar tertiary structures but totally different targets. Actin-specific ARTs are the A components of binary toxins, ADP-ribosylate actin at Arg177, leading to the depolymerization of the actin cytoskeleton. On the other hand, C3-like exoenzymes are Rho-specific ARTs, ADP-ribosylate Rho GTPases at Asn41, exerting an indirect effect on the actin cytoskeleton. This review focuses on the differences and similarities of actin- and Rho-specific ARTs, especially with respect to their substrate recognition and cell entry mechanisms, based on structural studies.

Anabaena sp. DyP-type peroxidase is a tetramer consisting of two asymmetric dimers.

DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.

Conformational plasticity is crucial for C3-RhoA complex formation by ARTT-loop.

ADP-ribosylation is an important post-translational protein modification catalyzed by bacterial toxins and eukaryotic endogenous ADP-ribosyltransferases. Bacterial binary toxins and C3-like toxins recognize and ADP-ribosylate actin Arg177 and RhoA Asn41, respectively. Structural and mutational studies have identified an ADP-ribosylating turn-turn loop (ARTT-loop) that has been implicated in substrate specificity and recognition, although it has not been verified. Recently, we determined the crystal structure of the C3 exoenzyme-RhoA complex. The complex structure shows how C3 recognizes Rho GTPase and provides the first structural evidence for RhoA recognition by the ARTT-loop. The complex formation mediated by the ARTT-loop is through the intrinsic plasticity of C3 and RhoA. C3 changes the conformations of both the phosphate nicotinamide-loop and the ARTT-loop by NAD(+) and RhoA binding, respectively. In contrast, RhoA changes the conformations of switch I and II regions upon C3 binding with a particular conformation, irrespective of the bound nucleotide (GTP or GDP).

Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure.

C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD(+) to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3.

Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria.

Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP. We also show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its homologs form a novel family of subtilases having an external chaperone.

Reaction Mechanism of Mono-ADP-Ribosyltransferase Based on Structures of the Complex of Enzyme and Substrate Protein.

Mono-ADP-ribosylation is a post-translational protein modification catalyzed by bacterial toxins and exoenzymes that function as ADP-ribosyltransferases. Despite the importance of this modification, the reaction mechanism remains poorly understood due to a lack of information on the crystal structure of these enzymes in complex with a substrate protein. Recently, the structures of two such complexes became available, which shed new light on the mechanisms of mono-ADP-ribosylation. In this review, we consider the reaction mechanism based on the structures of ADP-ribosyltransferases in complex with a substrate protein.

Substrate selectivity of bacterial monoacylglycerol lipase based on crystal structure.

Lipases, which are conserved from bacteria to mammals, catalyze the hydrolysis of acylglycerol to free fatty acids and glycerol. Monoacylglycerol lipase (MGL) specifically catalyzes the hydrolysis of monoacylglycerol. Although there have been numerous studies of the structure of lipases, there have been few studies of MGL. Here, we report the crystal structure of authentic MGL isolated from Bacillus sp. H257 (bMGL). The crystal diffracts to 1.96 Å resolution. It belongs to space group P21212, and the unit cell parameters are a=99.7 Å, b=106.1 Å and c=43.0 Å. As in other lipases, three structural features for lipase activity are conserved in bMGL: the glycine-X-serine-X-glycine motif, catalytic triad and cap region. The structure of bMGL appears to be closed, as the cap region covers the active site entrance. The isolated bMGL hydrolyzed 2-AG, a known human MGL-specific substrate. Based on a 2-AG bound model, we discuss the substrate selectivity. The functional and structural features of bMGL provide insight how its substrate selectivity is determined and how specific inhibitors of bacterial MGL could be designed, which may be useful for development of novel antibiotics.

Crystallization and preliminary X-ray diffraction studies of a surface mutant of the middle domain of PB2 from human influenza A (H1N1) virus.

In the last hundred years, four influenza pandemics have been experienced, beginning with that in Spain in 1918. Influenza A virus causes severe pneumonia and its RNA polymerase is an important target for drug design. The influenza A (H1N1) virus has eight ribonucleoprotein complexes, which are composed of viral RNA, RNA polymerases and nucleoproteins. PB2 forms part of the RNA polymerase complex and plays an important role in binding to the cap structure of host mRNA. The middle domain of PB2 includes a cap-binding site. The structure of PB2 from H1N1 complexed with m(7)GTP has not been reported. Plate-like crystals of the middle domain of PB2 from H1N1 were obtained, but the quality of these crystals was not good. An attempt was made to crystallize the middle domain of PB2 complexed with m(7)GTP using a soaking method; however, electron density for m(7)GTP was not observed on preliminary X-ray diffraction analysis. This protein has hydrophobic residues on its surface and is stable in the presence of high salt concentrations. To improve the solubility, a surface double mutant (P453H and I471T) was prepared. These mutations change the surface electrostatic potential drastically. The protein was successfully prepared at a lower salt concentration and good cube-shaped crystals were obtained using this protein. Here, the crystallization and preliminary X-ray diffraction analysis of this mutant of the middle domain of PB2 are reported.

Conformational polymorphism of m7GTP in crystal structure of the PB2 middle domain from human influenza A virus.

Influenza pandemics with human-to-human transmission of the virus are of great public concern. It is now recognized that a number of factors are necessary for human transmission and virulence, including several key mutations within the PB2 subunit of RNA-dependent RNA polymerase. The structure of the middle domain in PB2 has been revealed with or without m(7)GTP, thus the middle domain is considered to be novel target for structure-based drug design. Here we report the crystal structure of the middle domain of H1N1 PB2 with or without m(7)GTP at 1.9 Å and 2.0 Å resolution, respectively, which has two mutations (P453H, I471T) to increase electrostatic potential and solubility. Here we report the m(7)GTP has unique conformation differ from the reported structure. 7-methyl-guanine is fixed in the pocket, but particularly significant change is seen in ribose and triphosphate region: the buried 7-methyl-guanine indeed binds in the pocket forming by H357, F404, E361 and K376 but the triphosphate continues directly to the outer domain. The presented conformation of m(7)GTP may be a clue for the anti-influenza drug-design.

Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex.

Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD(+) analog ßTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD(+)-bound form (NAD(+)-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD(+)-Ia-actin complex. Here we report high-resolution structures of NAD(+)-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD(+) under different conditions. The structures of NAD(+)-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the ßTAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases.