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.

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.

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.

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.

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.

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.

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.

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.

General ADP-Ribosylation Mechanism Based on the Structure of ADP-Ribosyltransferase-Substrate Complexes.

ADP-ribosylation is a ubiquitous modification of proteins and other targets, such as nucleic acids, that regulates various cellular functions in all kingdoms of life. Furthermore, these ADP-ribosyltransferases (ARTs) modify a variety of substrates and atoms. It has been almost 60 years since ADP-ribosylation was discovered. Various ART structures have been revealed with cofactors (NAD+ or NAD+ analog). However, we still do not know the molecular mechanisms of ART. It needs to be better understood how ART specifies the target amino acids or bases. For this purpose, more information is needed about the tripartite complex structures of ART, the cofactors, and the substrates. The tripartite complex is essential to understand the mechanism of ADP-ribosyltransferase. This review updates the general ADP-ribosylation mechanism based on ART tripartite complex structures.

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.

Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8.

Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.

Role of side-edge site of sphingomyelinase from Bacillus cereus.

Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.

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.

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin.

The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.

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.

Structural basis for the kexin-like serine protease from Aeromonas sobria as sepsis-causing factor.

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 A resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs.

Refolding, characterization and crystal structure of (S)-malate dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix.

Tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon Aeropyrum pernix, and the enzyme was identified as (S)-malate dehydrogenase (MDH), which, when produced in Escherichia coli, was mainly obtained as an inactive inclusion body. The inclusion body was dissolved in 6 M guanidine-HCl and gradually refolded to the active enzyme through dilution of the denaturant. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of about 110 kDa. NADP was preferred as a coenzyme over NAD for (S)-malate oxidation and, unlike MDHs from other sources, this enzyme readily catalyzed the oxidation of (2S,3S)-tartrate and (2S,3R)-tartrate. The tartrate oxidation activity was also observed in MDHs from the hyperthermophilic archaea Methanocaldococcus jannaschii and Archaeoglobus fulgidus, suggesting these hyperthermophilic MDHs loosely bind their substrates. The refolded A. pernix MDH was also crystallized, and the structure was determined at a resolution of 2.9 A. Its overall structure was similar to those of the M. jannaschii, Chloroflexus aurantiacus, Chlorobium vibrioforme and Cryptosporidium parvum [lactate dehydrogenase-like] MDHs with root-mean-square-deviation values between 1.4 and 2.1 A. Consistent with earlier reports, Ala at position 53 was responsible for coenzyme specificity, and the next residue, Arg, was important for NADP binding. Structural comparison revealed that the hyperthermostability of the A. pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme.

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.