Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G(734). Our structures provide fundamental insights into the interactions between the activase and the G(734) loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G(734) of pyruvate formate-lyase.     

Structural basis for ligand and substrate recognition by torovirus hemagglutinin esterases

Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona- and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-O-acetylesterase domains almost identical to corresponding domains in orthomyxo- and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine–Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptor-binding site seems to have been designed to use 9-mono- and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins.     

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5′ to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5′ to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2′-hydroxyl of the nucleotide 5′ to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.The twister RNA motif was identified by bioinformatic searches and then validated biochemically to be a small self-cleaving ribozyme (1). This recently discovered class of ribozymes is called twister because its conserved secondary structure resembles the ancient Egyptian hieroglyph “twisted flax.” Representatives of the twister ribozyme class are found in all domains of life, but its biological role has yet to be determined. In addition to twister, the small self-cleaving ribozyme family includes the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozymes (the glmS ribozyme is upsteam of the the glmS gene that codes for the enzyme that catalyzes glucosamine-6-phosphate production) (1–6).The small self-cleaving ribozyme family can be split into two groups based on whether their active site is formed by an irregular helix (hammerhead, hairpin, and VS) or a double pseudoknot (PK) structure (HDV and glmS) (7). The structures of HDV and glmS are known, whereas twister was predicted from representative sequences to use two PKs to form its active site. It was expected that twister would be smaller in size than either HDV or glmS and more comparable in size and complexity to hammerhead (1).The self-cleavage rate constant of twister is estimated to be as rapid as or slightly more rapid than the hammerhead ribozyme. The estimated rate constants (k_obs) for twister is 1,000 per minute, and the experimental k_obs for hammerhead is 870 per minute (1, 8). These two ribozymes are ∼100- to 500-fold faster than other small self-cleaving ribozymes (2–10 per minute under the similar in vitro reaction conditions) (1, 8–10). Twister constructs previously tested exhibited a maximum cleavage rate at 1 mM Mg²⁺ and pH 7.4 (1). However, twister does not require magnesium or other divalent cations for catalysis; thus, magnesium is important only for structure formation (1).Biochemical in-line probing experiments and bioinformatics suggested that the consensus secondary structure of twister contains three to six stems, of which P1, P2, and P4 are required, whereas P0, P3, and P5 are optional; that the RNA can be circularly permutated; and that it contains internal and terminal loops that form two PKs (1, 11). Mutations in any of the highly conserved nucleotides or mutations that disrupt the P1 stem, P2 stem, P4 stem, or the two PKs significantly decrease the catalytic rate (1). Other mutational analysis indicated that several nucleotides are important for self-cleavage, but these nucleotides were not expected to be involved in canonical Watson–Crick (WC) base pairing or displayed any covariation (1).All small self-cleaving ribozymes undergo a specific internal transesterification reaction in which the ribose 2′-oxygen, phosphorus, and 5′-ribose oxygen are aligned for an S_N2-like reaction, yielding products with a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. This single-step reaction is analogous to the reaction catalyzed by RNase A, except that the protein enzyme undergoes a second step to remove the cyclic phosphate (12). There are four general strategies contributing to RNA self-cleavage via internal phosphoester transfer: (i) orientation of the reactive atoms for in-line nucleophilic attack; (ii) neutralization of the negative charge on the nonbridging oxygen atoms of the cleavage site phosphate; (iii) deprotonation of the 2′ oxygen nucleophile; and (iv) neutralization of the developing negative charge on the 5′ leaving group (9). First, all small self-cleaving ribozymes likely use the first strategy for phosphoester transfer, including twister (1, 9). Second, a part of the rate constant enhancement of twister is likely due to the base catalysis because the rate constant has a pH dependency suggesting that the shifts the pK_A of the 2′-hydroxyl group at the cleavage site (1, 9). However, it is unknown whether twister uses the transition-state stabilization and/or general acid strategies (1, 9).Here, we present the 4.1-Å resolution crystal structure of a twister ribozyme sequence from an organism found in the environment that has not been isolated of likely of prokaryotic origin (twister A) and the 3.1-Å resolution X-ray crystal structure of an Orzyza sativa (twister B) twister ribozyme in which the scissile phosphate and nucleotide 5′ to the cleavage site are ordered in the first and disordered in the second. These two crystal structures provide insights into twister’s catalytic mechanism and structural motifs used for formation of its active site. We identify groups that are involved in general base catalysis, transition state stabilization, and provide information about tertiary interactions that form the active site of twister. These structures show variations about how an RNA achieves site-specific self-cleavage and suggest a physical basis for how twister is able to rapidly self-cleave.     

Molecular hijacking of siroheme for the synthesis of heme and d1 heme

Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B(12), coenzyme F(430), and heme d(1) underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d(1) heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d(1) heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.     

Structural basis of sequence-specific collagen recognition by SPARC

Protein interactions with the collagen triple helix play a critical role in collagen fibril formation, cell adhesion, and signaling. However, structural insight into sequence-specific collagen recognition is limited to an integrin-peptide complex. A GVMGFO motif in fibrillar collagens (O denotes 4-hydroxyproline) binds 3 unrelated proteins: von Willebrand factor (VWF), discoidin domain receptor 2 (DDR2), and the extracellular matrix protein SPARC/osteonectin/BM-40. We report the crystal structure at 3.2 Å resolution of human SPARC bound to a triple-helical 33-residue peptide harboring the promiscuous GVMGFO motif. SPARC recognizes the GVMGFO motifs of the middle and trailing collagen chains, burying a total of 720 Ų of solvent-accessible collagen surface. SPARC binding does not distort the canonical triple helix of the collagen peptide. In contrast, a critical loop in SPARC is substantially remodelled upon collagen binding, creating a deep pocket that accommodates the phenylalanine residue of the trailing collagen chain (“Phe pocket”). This highly restrictive specificity pocket is shared with the collagen-binding integrin I-domains but differs strikingly from the shallow collagen-binding grooves of the platelet receptor glycoprotein VI and microbial adhesins. We speculate that binding of the GVMGFO motif to VWF and DDR2 also results in structural changes and the formation of a Phe pocket.     

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.Although the ubiquitin proteasome pathway plays essential roles in eukaryotes (reviewed in refs. 1 and 2), most bacterial species do not have proteasome systems and instead degrade proteins using ATP-dependent proteases like ClpP, Lon, and HslUV (reviewed in refs. 3 and 4). However, bacteria of the orders Actinomycetales and Nitrospirales also encode proteasomes that are structurally highly similar to eukaryotic and archaeal proteasomes (reviewed in refs. 5 and 6). Importantly, the human pathogen Mycobacterium tuberculosis (Mtb), an Actinomycete, requires proteasomal function to cause lethal infections in mice (7). Ablation of proteasomal degradation sensitizes bacteria to nitric oxide, an antimicrobial free radical made by macrophages and other cell types, and attenuates bacterial growth in mice (7–9). The potential to target persistent or latent bacteria has made the Mtb proteasome system a prioritized target for the development of antituberculosis drugs (10, 11). Indeed, Mtb-specific proteasome inhibitors have been identified that may provide a promising lead for new drugs to treat tuberculosis (12, 13).There are numerous similarities and differences between eukaryotic and bacterial proteasomes. The 20S proteasome core particle (20S CP), which consists of two seven-membered β-rings between two seven-membered α-rings, is highly conserved structurally between prokaryotes and eukaryotes (14–16). However, the accessory factors that associate with the 20S CPs quickly diverge among the domains of life. Both bacteria and eukaryotes use a covalent small protein modification to mark substrate proteins for degradation; however, the eukaryotic ubiquitin tag is a well-folded protein whereas the Mtb Pup (prokaryotic ubiquitin-like protein) tag is intrinsically disordered (17, 18). Furthermore, degradation of ubiquitylated proteins by eukaryotic 20S CPs largely relies on a complex regulatory particle that caps one or both ends of the 20S CP and includes a heterohexameric ring of adenosine triphosphatases (ATPases) for substrate recognition and unfolding (reviewed in refs. 19 and 20). In contrast, the mycobacterial 20S CP uses a homohexameric ATPase ring called Mpa (mycobacterial proteasome ATPase) for both the recognition and unfolding of pupylated proteins (18, 21, 22).In addition to the ATPase activators, proteolysis by eukaryotic proteasomes can also be stimulated by several ATP-independent factors, such as the 11S activators PA26 and PA28, as well as Blm10 (23–28). We and another group recently discovered that Mtb has an analogous factor encoded by Rv3780 that we call PafE (proteasome accessory factor E; also known as Bpa for bacterial proteasome activator), which stimulates the degradation of small peptides and β-casein in vitro (29, 30). Both studies also showed that a carboxyl (C)-terminal glycine-glutamine-tyrosine-leucine (GQYL) motif is essential for interacting with and activating 20S CPs, and the penultimate tyrosine residue contributes to activation similarly to tyrosines observed in the “HbYX” (hydrophobic-tyrosine-any amino acid) motif in other characterized proteasome activators (reviewed in ref. 28). Our work further showed that PafE promotes the degradation of at least one native Mtb protein substrate, heat-shock protein repressor (HspR), and that an Mtb pafE mutant is sensitive to heat shock and is attenuated for growth in mice (30). Importantly, PafE-mediated degradation does not require pupylation. Thus, there appear to be at least two independent paths for targeting proteins to the mycobacterial proteasome for degradation.Like the eukaryotic 11S proteasome activators, PafE does not require ATP to stimulate proteolysis. However, it was unknown if PafE formed heptameric complexes like PA26 or PA28. In this work, we show that PafE monomers assume a four-helix bundle structure that is similar to that found in 11S activators, but assemble differently into an unprecedented dodecameric ring structure with 12-fold symmetry. We used isothermal titration calorimetry, cryo-electron microscopy (cryo-EM), and X-ray crystallography to analyze interactions between PafE and 20S core particles, and found that PafE binding induces a larger gate-opening change than has been described for other organisms. We also found that PafE has an extended C terminus that limits the ability of PafE to activate proteasomal degradation in vitro and in vivo.     

Structural basis of ATG3 recognition by the autophagic ubiquitin-like protein ATG12

The autophagic ubiquitin-like protein (ublp) autophagy-related (ATG)12 is a component of the ATG12∼ATG5–ATG16L1 E3 complex that promotes lipid conjugation of members of the LC3 ublp family. A role of ATG12 in the E3 complex is to recruit the E2 enzyme ATG3. Here we report the identification of the ATG12 binding sequence in the flexible region of human ATG3 and the crystal structure of the minimal E3 complexed with the identified binding fragment of ATG3. The structure shows that 13 residues of the ATG3 fragment form a short β-strand followed by an α-helix on a surface area that is exclusive to ATG12. Mutational analyses of ATG3 confirm that four residues whose side chains make contacts with ATG12 are important for E3 interaction as well as LC3 lipidation. Conservation of these four critical residues is high in metazoan organisms and plants but lower in fungi. A structural comparison reveals that the ATG3 binding surface on ATG12 contains a hydrophobic pocket corresponding to the binding pocket of LC3 that accommodates the leucine of the LC3-interacting region motif. These findings establish the mechanism of ATG3 recruitment by ATG12 in higher eukaryotes and place ATG12 among the members of signaling ublps that bind liner sequences.Autophagy is a bulk degradation/recycling process essential for quality control in eukaryotic cells (1, 2). During autophagy, portions of cytoplasm are sequestered into membrane-bound vesicles called autophagosomes and transported into lysosomes for degradation. Autophagosome formation is mediated by a concerted action of a number of conserved autophagy-related (ATG) proteins (2, 3). The two classes of ubiquitin-like proteins (ublps) among these are members of the LC3 family in mammals or Atg8 in yeast and the conserved ATG12, which share some sequence similarities (4). LC3 and ATG12 are activated by the same E1 enzyme, ATG7, and then transferred to different E2 enzymes, ATG3 and ATG10, respectively. ATG3 conjugates LC3 to a lipid molecule, phosphatidylethanolamine (PE), on autophagosomal membranes. PE-conjugated LC3 plays crucial roles in control of membrane dynamics during autophagosome formation as well as in recruitment of cargos, such as aberrant proteins and damaged organelles, through binding to receptor proteins carrying an LC3-interacting region (LIR) motif (5). On the other hand, ATG10 attaches ATG12 to a structural protein, ATG5, and the resulting conjugate ATG12∼ATG5 acts like an E3 factor by stimulating the transfer of LC3 from ATG3 to PE (6). ATG12∼ATG5 exists as a complex with the coiled-coil protein ATG16L1 that localizes on autophagosome precursor membranes, resulting in LC3 lipidation for autophagosome formation (7). We previously showed that E3 activity requires the native covalent linkage between ATG12 and ATG5 and a composite surface patch formed by the residues of both of these proteins, and that the interaction with the E2 ATG3 is mostly mediated by ATG12 (8). However, it remains unclear how E3 communicates with or recruits E2.Here we describe atomic details of the ATG3–ATG12 interaction based on our crystal structure of a human E2–E3 complex containing this interaction. The results from accompanying interaction analyses and cellular LC3 lipidation assays support the importance of this interaction in LC3 lipidation and autophagy. We also show that the ATG3–ATG12 and LIR–LC3 interactions share some structural features. This unexpected finding together with our experimental data suggests that ATG12 is a protein recruitment apparatus in autophagy.     

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved Lewis^X binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam₂CSK₄ effectively, yet allows SSL3 to bind to an already formed TLR2–Pam₂CSK₄ complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2–lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.In recent years, Staphylococcus aureus has become a major health threat to both humans and domestic animals. It is found as a commensal bacterium in ∼30% of the human population, but when it becomes infectious it can cause a wide diversity of diseases, ranging from mild skin infections to life-threatening invasive conditions such as pneumonia and sepsis (1). Increased antibiotic resistance and a high amount of virulence factors secreted by S. aureus contribute to its emergence as a pathogen. Among these secreted virulence factors are the staphylococcal superantigen-like proteins (SSLs), a family of 14 proteins located on two genomic clusters (2–4). Recently, we and others identified SSL3 as a potent inhibitor of Toll-like receptor 2 (TLR2) (5, 6), an innate immunity receptor that is a dominant factor in immune recognition of S. aureus (7–10).TLR2 belongs to a family of 10 homologous innate immunity receptors that are activated by pathogen-associated molecular patterns (PAMPs) (11). TLR2 binds bacterial lipopeptides and lipoproteins. Subsequent formation of heterodimers with TLR1 or TLR6 leads to MyD88-dependent activation of the NF-κB pathway (12). TLR2 has dual ligand specificity that is determined by its dimerization partner; stimulation by diacyl lipopeptides from Gram-positive bacteria, including S. aureus, induces the formation of heterodimers with TLR6 (13), whereas triacyl lipopeptides from Gram-negative bacteria initiate formation of TLR2–TLR1 dimers (14). The structural basis for lipopeptide specificity was revealed by crystal structures of TLR2–TLR1 and TLR2–TLR6 complexes with their respective lipopeptide analogs Pam₃CSK₄ and Pam₂CSK₄: TLR2 binds two lipid tails in a large hydrophobic pocket, whereas the third lipid tail of triacyl lipopeptides is accommodated by a smaller pocket present in TLR1, but not in TLR6 (15, 16).The family of SSL proteins, including SSL3, share structural similarities to superantigens, but lack superantigenic activity. Interestingly, the functions that have been discovered for SSLs so far have all been linked to immune evasion. SSL5 inhibits neutrophil extravasation (17, 18) and phagocyte function (19, 20), SSL7 binds IgA and inhibits complement (21), and SSL10 inhibits IgG1-mediated phagocytosis (22, 23), blood coagulation (24), and the chemokine receptor CXCR4 (25). In addition to SSL3, also weak TLR2 inhibitory activity was observed for SSL4 (5), but it remains unknown whether that is its dominant function. This variety of immunomodulatory molecules and functions reflects the importance of the different components of our innate immune system in the defense against S. aureus (26).In this study we determined the crystal structures of SSL3 and the SSL3–TLR2 complex. In combination with mutagenesis and binding studies, our data provide a novel working mechanism of a functional TLR2 antagonist.     

Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog l-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the l-proline and l-arginine metabolism by acetylating l-Δ¹-pyrroline-5-carboxylate, leading to the l-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-l-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent k_cat value. The replacement of Asn135 led to a remarkable increase in the apparent K_m value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the l-Δ¹-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.     

Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination

Manipulation of the host’s ubiquitin network is emerging as an important strategy for counteracting and repurposing the posttranslational modification machineries of the host by pathogens. Ubiquitin E3 ligases encoded by infectious agents are well known, as are a variety of viral deubiquitinases (DUBs). Bacterial DUBs have been discovered, but little is known about the structure and mechanism underlying their ubiquitin recognition. In this report, we found that members of the Legionella pneumophila SidE effector family harbor a DUB module important for ubiquitin dynamics on the bacterial phagosome. Structural analysis of this domain alone and in complex with ubiquitin vinyl methyl ester (Ub-VME) reveals unique molecular contacts used in ubiquitin recognition. Instead of relying on the Ile44 patch of ubiquitin, as commonly used in eukaryotic counterparts, the SdeA_Dub module engages Gln40 of ubiquitin. The architecture of the active-site cleft presents an open arrangement with conformational plasticity, permitting deubiquitination of three of the most abundant polyubiquitin chains, with a distinct preference for Lys63 linkages. We have shown that this preference enables efficient removal of Lys63 linkages from the phagosomal surface. Remarkably, the structure reveals by far the most parsimonious use of molecular contacts to achieve deubiquitination, with less than 1,000 Ų of accessible surface area buried upon complex formation with ubiquitin. This type of molecular recognition appears to enable dual specificity toward ubiquitin and the ubiquitin-like modifier NEDD8.Ubiquitin, a small, 76-aa protein modifier, is involved in a wide array of eukaryotic cellular processes. The functionality of ubiquitin depends on the precise timing of the conjugation/deconjugation of the C terminus of ubiquitin to the ε-amino group of a lysine residue of a target protein. At the heart of this process are ligases (responsible for the covalent attachment of ubiquitin) and deubiquitinases (DUBs), which function to cleave isopeptide bonds between ubiquitin and substrates or within polyubiquitin chains (1). Even though many eukaryotic DUBs have already been characterized, little is known of these enzymes in prokaryotes (1–3).Given the essential role of ubiquitination in eukaryotic cells, it is not surprising that infectious agents have evolved numerous elegant strategies to exploit host signaling mediated by ubiquitination. Many bacterial pathogens use virulence factors to hijack the host ubiquitin pathway to establish successful infections (4). Even though E3 ubiquitin ligases of bacterial or viral origin have been relatively well characterized, bacterial DUBs have not, despite their importance in the life cycles and pathogenicity of several microbial species, including Salmonella typhimurium (SseL), Chlamydia trachomatis (ChlaDub1 and ChlaDub2), and ElaD (Escherichia coli) (5–7). These bacterial DUBs belong to a larger group of peptidases called the CE clan (the MEROPS database), which comprises eukaryotic, bacterial, and viral representatives (7, 8). Although quite different from eukaryotic DUBs (<10% identity), bacterial DUBs are phylogenetically related to mammalian desumoylating enzymes (SENP1, 2, and 3) and the deneddylase Den1 (NEDP1 or SENP8). In light of a large divergence from their eukaryotic counterparts, the overall structure of these proteases, as well as how they function and recognize ubiquitin to act as DUBs, remains to be structurally analyzed.Modulation of host ubiquitination pathways has emerged as an important theme in the pathogenesis of the opportunistic pathogen Legionella pneumophila responsible for Legionnaires’ disease (4, 9). Ubiquitinated species are enriched on the L. pneumophila-containing vacuole (LCV), and interference with such association disturbs bacterial intracellular replication (10). Among the approximately 300 effectors injected into host cells by L. pneumophila via the Dot/Icm type IV secretion system (11), eight proteins appear to possess F-box or U-box domains typical of some E3 ligases (12–16). This ligase activity has been demonstrated for LegU1, LegAU13/AnkB, and LubX (14, 17). A recent study revealed that SidC and SdcA are E3 ligases that catalyze the ligation reaction with a unique mechanism, and are required for efficient enrichment of ubiquitinated species on the bacterial phagosome (18).Because balanced regulation of host cell processes is critical for the virulence of L. pneumophila (19), we initiated experiments to identify L. pneumophila proteins with DUB activity. Our efforts revealed that members of the SidE family contain a DUB domain, which catalyzes the reaction with a Cys-His-Asp (CHD) catalytic triad showing a preference for Lys63-linked polyubiquitin chains. Structural analysis of the DUB domain and its complex with the mechanism-based inhibitor, ubiquitin vinyl methyl ester (Ub-VME), revealed a canonical core ubiquitin-like protease (Ulp) fold with a ubiquitin interface that is quite different from those used by structurally characterized eukaryotic DUBs. We also found that although the DUB activity is dispensable for the SidE family’s role in intracellular bacterial replication, it is important for the dynamics of the association of ubiquitinated species with the bacterial phagosome.     

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1–3 and 7–8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4–6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.     

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

The thiamin- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from E. coli catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO₂ and acetate. Concomitant reduction of the enzyme-bound flavin triggers membrane binding of the C terminus and shuttling of 2 electrons to ubiquinone 8, a membrane-bound mobile carrier of the electron transport chain. Binding to the membrane in vivo or limited proteolysis in vitro stimulate the catalytic proficiency by 2 orders of magnitude. The molecular mechanisms by which membrane binding and activation are governed have remained enigmatic. Here, we present the X-ray crystal structures of the full-length enzyme and a proteolytically activated truncation variant lacking the last 23 C-terminal residues inferred as important in membrane binding. In conjunction with spectroscopic results, the structural data pinpoint a conformational rearrangement upon activation that exposes the autoinhibitory C terminus, thereby freeing the active site. In the activated enzyme, Phe-465 swings into the active site and wires both cofactors for efficient electron transfer. The isolated C terminus, which has no intrinsic helix propensity, folds into a helical structure in the presence of micelles.     

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.     

Structural insight into the reaction mechanism and evolution of cytokinin biosynthesis

The phytohormone cytokinin regulates plant growth and development. This hormone is also synthesized by some phytopathogenic bacteria, such as Agrobacterium tumefaciens, and is as a key factor in the formation of plant tumors. The rate-limiting step of cytokinin biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferase (IPT). Agrobacterium IPT has a unique substrate specificity that enables it to increase trans-zeatin production by recruiting a metabolic intermediate of the host plant's biosynthetic pathway. Here, we show the crystal structures of Tzs, an IPT from A. tumefaciens, complexed with AMP and a prenyl-donor analogue, dimethylallyl S-thiodiphosphate. The structures reveal that the carbon-nitrogen-based prenylation proceeds by the SN2-reaction mechanism. Site-directed mutagenesis was used to determine the amino acid residues, Asp-173 and His-214, which are responsible for differences in prenyl-donor substrate specificity between plant and bacterial IPTs. IPT and the p loop-containing nucleoside triphosphate hydrolases likely evolved from a common ancestral protein. Despite structural similarities, IPT has evolved a distinct role in which the p loop transfers a prenyl moiety in cytokinin biosynthesis.     

Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB

Mucopolysaccharidosis III (MPS III) has four forms (A-D) that result from buildup of an improperly degraded glycosaminoglycan in lysosomes. MPS IIIB is attributable to the decreased activity of a lysosomal alpha-N-acetylglucosaminidase (NAGLU). Here, we describe the structure, catalytic mechanism, and inhibition of CpGH89 from Clostridium perfringens, a close bacterial homolog of NAGLU. The structure enables the generation of a homology model of NAGLU, an enzyme that has resisted structural studies despite having been studied for >20 years. This model reveals which mutations giving rise to MPS IIIB map to the active site and which map to regions distant from the active site. The identification of potent inhibitors of CpGH89 and the structures of these inhibitors in complex with the enzyme suggest small-molecule candidates for use as chemical chaperones. These studies therefore illuminate the genetic basis of MPS IIIB, provide a clear biochemical rationale for the necessary sequential action of heparan-degrading enzymes, and open the door to the design and optimization of chemical chaperones for treating MPS IIIB.     

Structural basis for alcohol modulation of a pentameric ligand-gated ion channel

Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the "linking tunnel." These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.     

Impaired vasodilation by red blood cells in sickle cell disease

Red blood cells (RBCs) have been ascribed a unique role in dilating blood vessels, which requires O2-regulated binding and bioactivation of NO by Hb and transfer of NO equivalents to the RBC membrane. Vasoocclusion in hypoxic tissues is the hallmark of sickle cell anemia. Here we show that sickle cell Hb variant S (HbS) is deficient both in the intramolecular transfer of NO from heme iron (iron nitrosyl, FeNO) to cysteine thiol (S-nitrosothiol, SNO) that subserves bioactivation, and in transfer of the NO moiety from S-nitrosohemoglobin (SNO-HbS) to the RBC membrane. As a result, sickle RBCs are deficient in membrane SNO and impaired in their ability to mediate hypoxic vasodilation. Further, the magnitudes of these impairments correlate with the clinical severity of disease. Thus, our results suggest that abnormal RBC vasoactivity contributes to the vasoocclusive pathophysiology of sickle cell anemia, and that the phenotypic variation in expression of the sickle genotype may be explained, in part, by variable deficiency in RBC processing of NO. More generally, our findings raise the idea that defective NO processing may characterize a new class of hemoglobinopathy.     

Structural basis for mouse receptor recognition by SARS-CoV-2 omicron variant

The sudden emergence and rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant has raised questions about its animal reservoir. Here, we investigated receptor recognition of the omicron’s receptor-binding domain (RBD), focusing on four of its mutations (Q493R, Q498R, N501Y, and Y505H) surrounding two mutational hotspots. These mutations have variable effects on the RBD’s affinity for human angiotensin-converting enzyme 2 (ACE2), but they all enhance the RBD’s affinity for mouse ACE2. We further determined the crystal structure of omicron RBD complexed with mouse ACE2. The structure showed that all four mutations are viral adaptations to mouse ACE2: three of them (Q493R, Q498R, and Y505H) are uniquely adapted to mouse ACE2, whereas the other one (N501Y) is adapted to both human ACE2 and mouse ACE2. These data reveal that the omicron RBD was well adapted to mouse ACE2 before omicron started to infect humans, providing insight into the potential evolutionary origin of the omicron variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant emerged abruptly and spread rapidly around the globe (1–4). Tracking the animal reservoir of SARS-CoV-2 and its variants is important for understanding the current COVID-19 pandemic and preventing future pandemics. Speculations about the source of the omicron variant are abundant, yet experimental evidence has been scarce (5). The interactions between the receptor-binding domain (RBD) of coronavirus spike proteins and their host receptor are among the best systems for understanding coronavirus evolution (6, 7). Both SARS-CoV-2 and closely related SARS-CoV-1 recognize human angiotensin-converting enzyme 2 (ACE2) as their receptor (8–10). Previous research on the receptor recognition of SARS-CoV-1 has provided insight into the animal origin of SARS-CoV-1 (11–15). The RBD of the original SARS-CoV-2 strain (i.e., prototypic RBD) differs from the RBD of a bat coronavirus by only a few residues, supporting a bat origin of the prototypic RBD (16). The omicron RBD (strain BA.2) differs from the prototypic RBD by 16 residues, seven of which are located in the receptor-binding motif (RBM) that directly contacts ACE2 (3). To recover the evolutionary traces left by these RBM mutations, this study compared the structural adaptations of the omicron RBD to ACE2 from human and mouse, two possible sources of omicron (5).Three virus-binding hotspots have been identified at the interfaces between SARS-CoV-2 RBD and human ACE2 (hACE2) and between SARS-CoV-1 RBD and hACE2 (14, 17, 18). These hotspots center on Lys31 in hACE2 (i.e., hotspot-31), Lys353 in hACE2 (i.e., hotspot-353), and a receptor-binding ridge in the viral RBD (i.e., hotspot-ridge) (Fig. 1A). These virus-binding hotspots are also mutational hotspots for SARS-CoV-1: all of the RBM mutations occurred around the hotspots and impacted the structural stability of the hotspots (13, 14). Establishment of the “hotspots” concept was instrumental in determining the molecular mechanisms by which SARS-CoV-1 was transmitted from palm civets to humans (11–15). The RBM mutations in the SARS-CoV-2 omicron variant are also around the hotspots (Fig. 1A). Curiously, only a few of these omicron mutations enhance the RBD’s affinity for hACE2, while some other mutations reduce it (Fig. 1B) (17). Structural details of the interface between the omicron RBM (strain BA.1) and hACE2 elucidated the role of each of these mutations in binding hACE2 (17). The omicron mutations that reduce the RBD’s affinity for hACE2 are structurally incompatible with hACE2, raising questions about what other species may have mediated the evolution of omicron.Open in a separate windowFig. 1.Binding interactions between SARS-CoV-2 RBD (from prototypic strain or omicron strain) and ACE2 (from human or mouse). (A) Structure of the interface between prototypic RBM and hACE2 (PDB ID: 6VW1). RBM is in magenta. hACE2 is in green. RBD residues that have undergone mutations from the prototypic strain to the omicron variant (strain BA.2) are shown as sticks. Three mutational hotspots are highlighted: hotspot-353 centers on Lys353 in hACE2, hotspot-31 centers on Lys31 in hACE2, and hotspot-ridge centers on the receptor-binding ridge in hACE2. (B and C) SPR assay for the binding of RBD (from prototypic strain or omicron strain) to ACE2 (from human or mouse). ACE2-Fc was coated to a protein A chip in a fixed direction, and individual RBDs flowed through. Data in B are from one of our recent studies (17), except that the omicron variant (strain BA.2) in this study replaced strain BA.1 in the previous study. Data in C are from the current study. The data in B and C are presented as mean ± SEM (n = 3 or n = 4) on a log scale. A Student’s two-tailed t test was performed to analyze the statistical difference between the RBD on the Left in either panel and each of the other RBDs in the same panel; the results are labeled on top of each bar. The statistical difference between the R493Q mutation and the N477S/R493Q double mutations was also analyzed in C; the result was labeled between the two bars. The horizontal dashed lines represent the measurements for the prototypic RBD in B or the omicron RBD in C and are used for comparison with other measurements in the respective panel. ***P < 0.001; **P < 0.01; *P < 0.05. n.s., statistically not significant, N.D., not detected.In this study, we provide biochemical and structural evidence demonstrating that the omicron mutations are better adapted to mouse ACE2 (mACE2) than to hACE2, suggesting that mice mediated the onset of the omicron variant. Our study helps clarify the animal reservoir of the omicron variant and contributes to the understanding of SARS-CoV-2 evolution. The findings may facilitate epidemiological surveillance of SARS-CoV-2 in animals to prevent future coronavirus pandemics.     

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr–sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.