Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

Crystal structures of YeiE from Cronobacter sakazakii and the role of sulfite tolerance in gram-negative bacteria.

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

Structural studies of a bacterial condensin complex reveal ATP-dependent disruption of intersubunit interactions.

Condensins are key mediators of chromosome condensation across organisms. Like other condensins, the bacterial MukBEF condensin complex consists of an SMC family protein dimer containing two ATPase head domains, MukB, and two interacting subunits, MukE and MukF. We report complete structural views of the intersubunit interactions of this condensin along with ensuing studies that reveal a role for the ATPase activity of MukB. MukE and MukF together form an elongated dimeric frame, and MukF's C-terminal winged-helix domains (C-WHDs) bind MukB heads to constitute closed ring-like structures. Surprisingly, one of the two bound C-WHDs is forced to detach upon ATP-mediated engagement of MukB heads. This detachment reaction depends on the linker segment preceding the C-WHD, and mutations on the linker restrict cell growth. Thus ATP-dependent transient disruption of the MukB-MukF interaction, which creates openings in condensin ring structures, is likely to be a critical feature of the functional mechanism of condensins.

Isocitrate binds to the itaconic acid-responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis.

Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain of RipR from the intracellular pathogen Salmonella. The RipR regulatory domain structure exhibited the typical dimeric arrangement with the putative ligand-binding site between the two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as the physiological ligand of RipR, whose intracellular level is increased in response to itaconic acid stress. We further found that 3-phenylpropionic acid significantly decreased the resistance of the bacteria to an itaconic acid challenge. Consistently, the complex structure revealed that the compound is antagonistically bound to the RipR ligand-binding site. This study provides the molecular basis of bacterial survival in itaconic acid stress from our immune systems. Further studies are required to reveal biochemical activity, which would elucidate how Salmonella survives in macrophage phagosomes by defending against itaconic acid inhibition of bacterial metabolism.

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae.

The bacterial second messenger bis-(3'-5')-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain-containing diguanylate cyclases and degraded by HD-GYP domain-containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain-containing PDE-As to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.

Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain.

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head-tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle-dependent regulation of nuclear morphology at the molecular level.

Structural Basis of the Inhibition of L-Methionine γ-Lyase from Fusobacterium nucleatum.

Fusobacterium nucleatum is a lesion-associated obligate anaerobic pathogen of destructive periodontal disease; it is also implicated in the progression and severity of colorectal cancer. Four genes (FN0625, FN1055, FN1220, and FN1419) of F. nucleatum are involved in producing hydrogen sulfide (H2S), which plays an essential role against oxidative stress. The molecular functions of Fn1419 are known, but their mechanisms remain unclear. We determined the crystal structure of Fn1419 at 2.5 Å, showing the unique conformation of the PLP-binding site when compared with L-methionine γ-lyase (MGL) proteins. Inhibitor screening for Fn1419 with L-cysteine showed that two natural compounds, gallic acid and dihydromyricetin, selectively inhibit the H2S production of Fn1419. The chemicals of gallic acid, dihydromyricetin, and its analogs containing trihydroxybenzene, were potentially responsible for the enzyme-inhibiting activity on Fn1419. Molecular docking and mutational analyses suggested that Gly112, Pro159, Val337, and Arg373 are involved in gallic acid binding and positioned close to the substrate and pyridoxal-5'-phosphate-binding site. Gallic acid has little effect on the other H2S-producing enzymes (Fn1220 and Fn1055). Overall, we proposed a molecular mechanism underlying the action of Fn1419 from F. nucleatum and found a new lead compound for inhibitor development.

Structural basis for the interaction between unfarnesylated progerin and the Ig-like domain of lamin A/C in premature aging disorders.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by C-terminally truncated lamin A, termed as the pre-progerin product. Progerin is a C-terminally farnesylated protein derived from pre-progerin, which causes nuclear deformation at the inner-nuclear membrane. As an alternative or additional mechanism, a farnesylation-independent abnormal interaction between the C-terminus of progerin and Ig-like domain has been proposed. However, the molecular mechanism underlying the role of unfarnesylated C-terminus of pre-progerin in HGPS remains largely unknown. In this study, we determined the crystal structures of C-terminal peptide of progerin and Ig-like domain of lamin A/C. Results showed that the C-terminal cysteine residue of progerin forms a disulfide bond with the only cysteine residue of the Ig-like domain. This finding suggested that unfarnesylated progerin can form a disulfide bond with the Ig-like domain in the lamin meshwork. The Alphafold2-assisted docking structure showed that disulfide bond formation was promoted by a weak interaction between the groove of Ig-like domain and the unfarnesylated C-terminal tail region of progerin. Our results provide molecular insights into the normal aging process as well as premature aging of humans.

Structural basis for HOCl recognition and regulation mechanisms of HypT, a hypochlorite-specific transcriptional regulator.

Hypochlorous acid (HOCl) is generated in the immune system to kill microorganisms. In Escherichia coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). Here, we identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, we identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand-binding site in the RD. Furthermore, we proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudopalindromic motifs that are separated by ∼100 bp, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms.

Anti-Inflammatory and Neuroprotective Effects of Morin in an MPTP-Induced Parkinson's Disease Model.

Neurodegenerative diseases such as Parkinson's disease (PD) are known to be related to oxidative stress and neuroinflammation, and thus, modulating neuroinflammation offers a possible means of treating PD-associated pathologies. Morin (2',3,4',5,7-pentahydroxy flavone) is a flavonol with anti-oxidative and anti-inflammatory effects found in wines, herbs, and fruits. The present study was undertaken to determine whether a morin-containing diet has protective effects in an MPTP-induced mouse model of PD. Mice were fed a control or morin diet for 34 days, and then MPTP (30 mg/kg, i.p.) was administered daily for 5 days to induce a PD-like pathology. We found that dietary morin prevented MPTP-induced motor dysfunction and ameliorated dopaminergic neuronal damage in striatum (STR) and substantia nigra (SN) in our mouse model. Furthermore, MPTP-induced neuroinflammation was significantly reduced in mice fed morin. In vitro studies showed that morin effectively suppressed glial activations in primary microglia and astrocytes, and biochemical analysis and a docking simulation indicated that the anti-inflammatory effects of morin were mediated by blocking the extracellular signal-regulated kinase (ERK)-p65 pathway. These findings suggest that morin effectively inhibits glial activations and has potential use as a functional food ingredient with therapeutic potential for the treatment of PD and other neurodegenerative diseases associated with neuroinflammation.

Structural and Functional Analysis of the Pyridoxal Phosphate Homeostasis Protein YggS from Fusobacterium nucleatum.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.

Structure and function of the hypochlorous acid-induced flavoprotein RclA from Escherichia coli.

In response to microbial invasion, the animal immune system generates hypochlorous acid (HOCl) that kills microorganisms in the oxidative burst. HOCl toxicity is amplified in the phagosome through import of the copper cation (Cu2+). In Escherichia coli and Salmonella, the transcriptional regulator RclR senses HOCl stress and induces expression of the RclA, -B, and -C proteins involved in bacterial defenses against oxidative stress. However, the structures and biochemical roles of the Rcl proteins remain to be elucidated. In this study, we first examined the role of the flavoprotein disulfide reductase (FDR) RclA in the survival of Salmonella in macrophage phagosomes, finding that RclA promotes Salmonella survival in macrophage vacuoles containing sublethal HOCl levels. To clarify the molecular mechanism, we determined the crystal structure of RclA from E. coli at 2.9 Å resolution. This analysis revealed that the structure of homodimeric RclA is similar to those of typical FDRs, exhibiting two conserved cysteine residues near the flavin ring of the cofactor flavin adenine dinucleotide (FAD). Of note, we observed that Cu2+ accelerated RclA-mediated oxidation of NADH, leading to a lowering of oxygen levels in vitro Compared with the RclA WT enzyme, substitution of the conserved cysteine residues lowered the specificity to Cu2+ or substantially increased the production of superoxide anion in the absence of Cu2+ We conclude that RclA-mediated lowering of oxygen levels could contribute to the inhibition of oxidative bursts in phagosomes. Our study sheds light on the molecular basis for how bacteria can survive HOCl stress in macrophages.

Beta-strand-mediated dimeric formation of the Ig-like domains of human lamin A/C and B1.

Lamins are nuclear intermediate filament proteins that play an essential role in maintaining the nuclear structure by forming a 3-D meshwork. Lamins consist of the N-terminal unstructured head, the coiled-coil rod domain, and the C-terminal tail, which is mostly unstructured except for the Ig-like domain. To date, the Ig-like domain has been characterized as a monomeric structure. Here, we determined the crystal structures of human lamin A/C, including the Ig-like domain and its N- and C-terminal flanking sequences. Interestingly, the structures showed a homodimer formed by beta-strand interactions between the N- and C-terminal flanking sequences. This interaction also provides a molecular implication for the creation of a 3-D meshwork between the 3.5-nm-thick filaments. Furthermore, we determined the crystal structure of the corresponding region of lamin B1. The structure showed a similar dimeric assembly, also formed by beta-strand interactions, albeit the intersubunit distance was much shorter. Since the Ig-like domain contains many genetic hotspots causing lamin-related diseases in lamin A/C, our findings will help understand the detailed assembly of lamins in a 3-D meshwork structure and lamin-related diseases at the molecular level.

High-resolution structures of annexin A5 in a two-dimensional array.

Annexins are soluble cytosolic proteins that bind to cell membranes. Annexin A5 self-assembles into a two-dimensional (2D) array and prevents cell rupture by attaching to damaged membranes. However, this process is not fully understood at the molecular level. In this study, we determined the crystal structures of annexin A5 with and without calcium (Ca2+) and confirmed the Ca2+-dependent outward motion of a tryptophan residue. Strikingly, the two structures exhibited the same crystal packing and 2D arrangement into a p3 lattice, which agrees well with the results of low-resolution structural imaging. High-resolution structures indicated that a three-fold interaction near the tryptophan residue is important for mediating the formation of the p3 lattice. A hypothesis on the promotion of p3 lattice formation by phosphatidyl serine (PS) is also suggested. This study provides molecular insight into how annexins modulate the physical properties of cell membranes as a function of Ca2+ concentration and the phospholipid composition of the membrane.

Crystal Structure of Aeromonas hydrophila Cytoplasmic 5'-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine (MTA/SAH) nucleosidase (MTAN) is an important enzyme in a number of critical biological processes. Mammals do not express MtaN, making this enzyme an attractive antibacterial drug target. In pathogen Aeromonas hydrophila, two MtnN subfamily genes (MtaN-1 and MtaN-2) play important roles in the periplasm and cytosol, respectively. We previously reported structural and functional analyses of MtaN-1, but little is known regarding MtaN-2 due to the lack of a crystal structure. Here, we determined the crystal structure of cytosolic A. hydrophila MtaN-2 in complex with adenine (ADE), which is a cleavage product of adenosine. AhMtaN-1 and AhMtaN-2 exhibit a high degree of similarity in the α-ß-α sandwich fold of the core structural motif. However, there is a structural difference in the nonconserved extended loop between ß7 and α3 that is associated with the channel depth of the substrate-binding pocket and dimerization. The ADE molecules in the substrate-binding pockets of AhMtaN-1 and AhMtaN-2 are stabilized with π-π stacking by Trp199 and Phe152, respectively, and the hydrophobic residues surrounding the ribose-binding sites differ. A structural comparison of AhMtaN-2 with other MtaN proteins showed that MtnN subfamily proteins exhibit a unique substrate-binding surface and dimerization interface.

Structural insight into the carboxylesterase BioH from Klebsiella pneumoniae.

The BioH carboxylesterase which is a typical α/ß-hydrolase enzyme involved in biotin synthetic pathway in most bacteria. BioH acts as a gatekeeper and blocks the further elongation of its substrate. In the pathogen Klebsiella pneumoniae, BioH plays a critical role in the biosynthesis of biotin. To better understand the molecular function of BioH, we determined the crystal structure of BioH from K. pneumoniae at 2.26 Šresolution using X-ray crystallography. The structure of KpBioH consists of an α-ß-α sandwich domain and a cap domain. B-factor analysis revealed that the α-ß-α sandwich domain is a rigid structure, while the loops in the cap domain shows the structural flexibility. The active site of KpBioH contains the catalytic triad (Ser82-Asp207-His235) on the interface of the α-ß-α sandwich domain, which is surrounded by the cap domain. Size exclusion chromatography shows that KpBioH prefers the monomeric state in solution, whereas two-fold symmetric dimeric formation of KpBioH was observed in the asymmetric unit, the conserved Cys31-based disulfide bonds can maintain the irreversible dimeric formation of KpBioH. Our study provides important structural insight for understanding the molecular mechanisms of KpBioH and its homologous proteins.

Crystal structure of the Siderophore-interacting protein SIP from Aeromonas hydrophila.

Siderophores acquire iron from hosts under iron-limiting conditions and play an essential role in the survival of microorganisms. Siderophore-interacting proteins (SIPs) from microbes release iron from the siderophore complex by reducing ferric iron to ferrous iron, but the molecular mechanism of iron reduction remains unclear. To better understand the molecular mechanism of SIPs, we herein report the crystal structure of Aeromonas hydrophila SIP (AhSIP) in complex with flavin adenine dinucleotide (FAD) as a cofactor. AhSIP consists of an N-terminal FAD binding domain and a C-terminal NADH binding domain, which are connected by a linker region. AhSIP showed unique structural differences in the orientation of the cofactor binding lobes when compared with SIP homologs. This study identified a cluster of three basic residues (Lys48, His259 and Arg262) in AhSIP distributed around a potential substrate binding pocket. In addition, AhSIP, containing the NADH binding motif E(L)VL-X3-GE, belongs to the group I subfamily. Our results show the diverse cofactor and substrate binding sites of the SIP family.

Structural and functional analyses of the lipase CinB from Enterobacter asburiae.

Lipases are widely present in various plants, animals and microorganisms, constituting a large category of enzymes. They have the ability to catalyze the cleavage of ester bonds. The lipase CinB from Enterobacter asburiae (E. asburiae) is an acetyl esterase. The primary amino acid sequence suggests that the EaCinB protein belongs to the α/ß-hydrolase (ABH) superfamily of the esterase/lipase superfamily. However, its molecular functions have not yet been determined. Here, we report the crystal structure of E. asburiae CinB at a 1.45 Šresolution. EaCinB contains a signal peptide, cap domain and catalytic domain. The active site of EaCinB contains the catalytic triad (Ser180-His307-Asp277) on the catalytic domain. The oxyanion hole is composed of Gly106 and Gly107 within the conserved sequence motif HGGG (amino acid residues 106-109). The substrate is accessible between the α1 and α2 helices or the α1 helix and catalytic domain. Narrow substrate pockets are formed by the α2 helix of the cap domain. Site-directed mutagenesis showed that EaCinB-W208H exhibits a higher catalytic ability than EaCinB-WT by approximately nine times. Our results provide insight into the molecular function of EaCinB.

Structural insight of the 5-(Hydroxyethyl)-methylthiazole kinase ThiM involving vitamin B1 biosynthetic pathway from the Klebsiella pneumoniae.

Thiamin pyrophosphate (TPP) is an essential co-factor in amino acid and carbohydrate metabolic pathways. The TPP-related vitamin B1 biosynthetic pathway is found in most bacterial, plant and lower eukaryotic processes; however, it is not present in humans. In bacterial thiamin synthesis and salvage pathways, the 5-(hydroxyethyl)-methylthiazole kinase (ThiM) is essential in the pathway forming TPP. Thus, ThiM is considered to be an attractive antibacterial drug target. Here, we determined the crystal structures of ThiM from pathogenic Klebsiella pneumoniae (KpThiM) and KpThiM in complex with its substrate 5-(hydroxyethyl)-4-methylthiazole (TZE). KpThiM, consisting of an α-ß-α domain, shows a pseudosymmetric trimeric formation. TZE molecules are located in the interface between the KpThiM subunits in the trimer and interact with Met49 and Cys200. Superimposition of the apo and TZE-complexed structures of KpThiM show that the side chains of the amino acids interacting with TZE and Mg2+ have a rigid configuration. Comparison of the ThiM structures shows that KpThiM could, in terms of sequence and configuration, be different from other ThiM proteins, which possess different amino acids that recognize TZE and Mg2+. The structures will provide new insight into the ThiM subfamily proteins for antibacterial drug development.

Biochemical and structural analysis of the Klebsiella pneumoniae cytidine deaminase CDA.

The emergence of drug-resistant strains of Klebsiella pneumoniae, has exacerbated the treatment and control of the disease caused by this bacterium. Cytidine deaminases (CDA) are zinc-dependent enzymes involved in the pyrimidine salvage pathway and catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. To illustrate the structural basis of CDA for a deeper knowledge of the molecular mechanisms underlying the salvage pathway, we reported here the biochemical and structural analysis of CDA from pathogenic K. pneumonia. KpCDA showed deaminase activity against cytidine as well as its analog cytarabine. The deaminase activity of KpCDA on cytarabine was 1.8 times higher than that on cytidine. KpCDA is composed of an N-terminal catalytic domain and a C-terminal noncatalytic domain. Zinc, which is involved in the activity of the catalytic domain, is coordinated by His102, Cys129, and Cys132, and two 1,4-dioxane molecules were present at the active sites. KpCDA exists as a dimer and shows distinct dimeric interface compared with other CDAs. Our results provide the structural features of KpCDA, and KpCDA might be a potential antibacterial target for the disease caused by K. pneumoniae.