Structural basis for differential activities of enantiomeric PPARγ agonists: Binding of S35 to the alternate site.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily. It functions as a ligand-activated transcription factor and plays important roles in the regulation of adipocyte differentiation, type 2 diabetes mellitus, and inflammation. Many PPARγ agonists bind to the canonical ligand-binding pocket near the activation function-2 (AF-2) helix (i.e., helix H12) of the ligand-binding domain (LBD). More recently, an alternate ligand-binding site was identified in PPARγ LBD; it is located beside the Ω loop between the helices H2' and H3. We reported previously that the chirality of two optimized enantiomeric PPARγ ligands (S35 and R35) differentiates their PPARγ transcriptional activity, binding affinity, and inhibitory activity toward Cdk5 (cyclin-dependent kinase 5)-mediated phosphorylation of PPARγ at Ser245 (in PPARγ1 numbering; Ser273 in PPARγ2 numbering). S35 is a PPARγ phosphorylation inhibitor with promising glucose uptake potential, whereas R35 behaves as a potent conventional PPARγ agonist. To provide a structural basis for understanding the differential activities of these enantiomeric ligands, we have determined crystal structures of the PPARγ LBD in complex with either S35 or R35. S35 and R35 bind to the PPARγ LBD in significantly different manners. The partial agonist S35 occupies the alternate site near the Ω loop, whereas the full agonist R35 binds entirely to the canonical LBP. Alternate site binding of S35 affects the PPARγ transactivation and the inhibitory effect on PPARγ Ser245 phosphorylation. This study provides a useful platform for the development of a new generation of PPARγ ligands as anti-diabetic drug candidates.

Crystal structure of Rv2258c from Mycobacterium tuberculosis H37Rv, an S-adenosyl-l-methionine-dependent methyltransferase.

The Mycobacterium tuberculosis Rv2258c protein is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase). Here, we have determined its crystal structure in three forms: a ligand-unbound form, a binary complex with sinefungin (SFG), and a binary complex with S-adenosyl-L-homocysteine (SAH). The monomer structure of Rv2258c consists of two domains which are linked by a long α-helix. The N-terminal domain is essential for dimerization and the C-terminal domain has the Class I MTase fold. Rv2258c forms a homodimer in the crystal, with the N-terminal domains facing each other. It also exists as a homodimer in solution. A DALI structural similarity search with Rv2258c reveals that the overall structure of Rv2258c is very similar to small-molecule SAM-dependent MTases. Rv2258c interacts with the bound SFG (or SAH) in an extended conformation maintained by a network of hydrogen bonds and stacking interactions. Rv2258c has a relatively large hydrophobic cavity for binding of the methyl-accepting substrate, suggesting that bulky nonpolar molecules with aromatic rings might be targeted for methylation by Rv2258c in M. tuberculosis. However, the ligand-binding specificity and the biological role of Rv2258c remain to be elucidated due to high variability of the amino acid residues defining the substrate-binding site.

The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of L,D-Carboxypeptidase.

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.

Structure of Csd3 from Helicobacter pylori, a cell shape-determining metallopeptidase.

Helicobacter pylori is associated with various gastrointestinal diseases such as gastritis, ulcers and gastric cancer. Its colonization of the human gastric mucosa requires high motility, which depends on its helical cell shape. Seven cell shape-determining genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5 and csd6) have been identified in H. pylori. Their proteins play key roles in determining the cell shape through modifications of the cell-wall peptidoglycan by the alteration of cross-linking or by the trimming of peptidoglycan muropeptides. Among them, Csd3 (also known as HdpA) is a bifunctional enzyme. Its D,D-endopeptidase activity cleaves the D-Ala(4)-mDAP(3) peptide bond between cross-linked muramyl tetrapeptides and pentapeptides. It is also a D,D-carboxypeptidase that cleaves off the terminal D-Ala(5) from the muramyl pentapeptide. Here, the crystal structure of this protein has been determined, revealing the organization of its three domains in a latent and inactive state. The N-terminal domain 1 and the core of domain 2 share the same fold despite a very low level of sequence identity, and their surface-charge distributions are different. The C-terminal LytM domain contains the catalytic site with a Zn(2+) ion, like the similar domains of other M23 metallopeptidases. Domain 1 occludes the active site of the LytM domain. The core of domain 2 is held against the LytM domain by the C-terminal tail region that protrudes from the LytM domain.

Mycobacterium tuberculosis Eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by Mtb, enhances survival of Mycobacterium smegmatis (Msm) in macrophages. Mtb Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through JNK-dependent inhibition of reactive oxygen species (ROS) generation. Mtb Eis was recently demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by Mtb Eis remains unanswered. Therefore, we have characterized both Mtb and Msm Eis proteins biochemically and structurally. We have discovered that Mtb Eis is an efficient N(ε)-acetyltransferase, rapidly acetylating Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, Msm Eis is more efficient as an N(α)-acetyltransferase. We also show that Msm Eis acetylates aminoglycosides as readily as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPS-induced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of Mtb Eis with a narrow channel seems more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of Msm Eis. We propose that Mtb Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and ROS generation by acetylating DUSP16/MKP-7. Our work thus provides insight into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by Mtb Eis.

Structural basis for the recognition of muramyltripeptide by Helicobacter pylori Csd4, a D,L-carboxypeptidase controlling the helical cell shape.

Helicobacter pylori infection causes a variety of gastrointestinal diseases, including peptic ulcers and gastric cancer. Its colonization of the gastric mucosa of the human stomach is a prerequisite for survival in the stomach. Colonization depends on its motility, which is facilitated by the helical shape of the bacterium. In H. pylori, cross-linking relaxation or trimming of peptidoglycan muropeptides affects the helical cell shape. Csd4 has been identified as one of the cell shape-determining peptidoglycan hydrolases in H. pylori. It is a Zn(2+)-dependent D,L-carboxypeptidase that cleaves the bond between the γ-D-Glu and the mDAP of the non-cross-linked muramyltripeptide (muramyl-L-Ala-γ-D-Glu-mDAP) of the peptidoglycan to produce the muramyldipeptide (muramyl-L-Ala-γ-D-Glu) and mDAP. Here, the crystal structure of H. pylori Csd4 (HP1075 in strain 26695) is reported in three different states: the ligand-unbound form, the substrate-bound form and the product-bound form. H. pylori Csd4 consists of three domains: an N-terminal D,L-carboxypeptidase domain with a typical carboxypeptidase fold, a central ß-barrel domain with a novel fold and a C-terminal immunoglobulin-like domain. The D,L-carboxypeptidase domain recognizes the substrate by interacting primarily with the terminal mDAP moiety of the muramyltripeptide. It undergoes a significant structural change upon binding either mDAP or the mDAP-containing muramyltripeptide. It it also shown that Csd5, another cell-shape determinant in H. pylori, is capable of interacting not only with H. pylori Csd4 but also with the dipeptide product of the reaction catalyzed by Csd4.

In what ways does placeness affect people's behavior? Focusing on personal place attachment and public place image as connecting parameter.

Placeness is believed to play a significant role in enhancing the well-being and place-use of individuals, contributing profoundly to how spaces are experienced and interacted with. Despite its perceived importance, there is ongoing debate and insufficient clarity about how exactly placeness influences people's behavior. This study aims to bridge this gap by theorizing and investigating the pathways from placeness to people's behavioral intentions, emphasizing the roles of personal place attachment and public place image as pivotal mediators in this relationship. To explore these dynamics, we conducted a survey in Japan, examining the complex interplay between placeness and behavioral intentions, given their rich cultural heritage and modern urban pressures. We employed the Partial Least Squares Structural Equation Modeling (PLS-SEM) approach for path analysis. The analysis suggests that placeness influences behavioral intention through personal place attachment. While placeness does affect public place image, this public image does not have an impact on behavioral intention. The results demonstrated that an individual's activities, experiences, and cognition of a place are significant factors in creating the intention to engage in word-of-mouth, recommendation, and revisiting behaviors. Policymakers, urban planners, and designers need to understand how to foster people's behavioral intentions when creating a place imbued with placeness.

Structural basis for the inhibition of Mycobacterium tuberculosis L,D-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains.

Difficulty in the treatment of tuberculosis and growing drug resistance in Mycobacterium tuberculosis (Mtb) are a global health issue. Carbapenems inactivate L,D-transpeptidases; meropenem, when administered with clavulanate, showed in vivo activity against extensively drug-resistant Mtb strains. LdtMt2 (Rv2518c), one of two functional L,D-transpeptidases in Mtb, is predominantly expressed over LdtMt1 (Rv0116c). Here, the crystal structure of N-terminally truncated LdtMt2 (residues Leu131-Ala408) is reported in both ligand-free and meropenem-bound forms. The structure of meropenem-inhibited LdtMt2 provides a detailed structural view of the interactions between a carbapenem drug and Mtb L,D-transpeptidase. The structures revealed that the catalytic L,D-transpeptidase domain of LdtMt2 is preceded by a bacterial immunogloblin-like Big_5 domain and is followed by an extended C-terminal tail that interacts with both domains. Furthermore, it is shown using mass analyses that meropenem acts as a suicide inhibitor of LdtMt2. Upon acylation of the catalytic Cys354 by meropenem, the `active-site lid' undergoes a large conformational change to partially cover the active site so that the bound meropenem is accessible to the bulk solvent via three narrow paths. This work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antituberculosis drugs against drug-resistant Mtb.

Structural and functional characterization of HP0377, a thioredoxin-fold protein from Helicobacter pylori.

Maturation of cytochrome c is carried out in the bacterial periplasm, where specialized thiol-disulfide oxidoreductases provide the correct reduction of oxidized apocytochrome c before covalent haem attachment. HP0377 from Helicobacter pylori is a thioredoxin-fold protein that has been implicated as a component of system II for cytochrome c assembly and shows limited sequence similarity to Escherichia coli DsbC, a disulfide-bond isomerase. To better understand the role of HP0377, its crystal structures have been determined in both reduced and partially oxidized states, which are highly similar to each other. Sedimentation-equilibrium experiments indicate that HP0377 is monomeric in solution. HP0377 adopts a thioredoxin fold but shows distinctive variations as in other thioredoxin-like bacterial periplasmic proteins. The active site of HP0377 closely resembles that of E. coli DsbC. A reductase assay suggests that HP0377 may play a role as a reductase in the biogenesis of holocytochrome c553 (HP1227). Binding experiments indicate that it can form a covalent complex with HP0518, a putative L,D-transpeptidase with a catalytic cysteine residue, via a disulfide bond. Furthermore, physicochemical properties of HP0377 and its R86A variant have been determined. These results suggest that HP0377 may perform multiple functions as a reductase in H. pylori.

High-resolution crystal structure of Streptococcus pyogenes ß-NAD⁺ glycohydrolase in complex with its endogenous inhibitor IFS reveals a highly water-rich interface.

One of the virulence factors produced by Streptococcus pyogenes is ß-NAD(+) glycohydrolase (SPN). S. pyogenes injects SPN into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. As SPN is toxic to bacterial cells themselves, S. pyogenes possesses the ifs gene that encodes an endogenous inhibitor for SPN (IFS). IFS is localized intracellularly and forms a complex with SPN. This intracellular complex must be dissociated during export through the cell envelope. To provide a structural basis for understanding the interactions between SPN and IFS, the complex was overexpressed between the mature SPN (residues 38-451) and the full-length IFS (residues 1-161), but it could not be crystallized. Therefore, limited proteolysis was used to isolate a crystallizable SPNct-IFS complex, which consists of the SPN C-terminal domain (SPNct; residues 193-451) and the full-length IFS. Its crystal structure has been determined by single anomalous diffraction and the model refined at 1.70 Å resolution. Interestingly, our high-resolution structure of the complex reveals that the interface between SPNct and IFS is highly rich in water molecules and many of the interactions are water-mediated. The wet interface may facilitate the dissociation of the complex for translocation across the cell envelope.

Crystal structures of Pseudomonas aeruginosa guanidinobutyrase and guanidinopropionase, members of the ureohydrolase superfamily.

Pseudomonas aeruginosa guanidinobutyrase (GbuA) and guanidinopropionase (GpuA) catalyze the hydrolysis of 4-guanidinobutyrate and 3-guanidinopropionate, respectively. They belong to the ureohydrolase superfamily, which includes arginase, agmatinase, proclavaminate amidinohydrolase, and formiminoglutamase. In this study, we have determined the crystal structures of GbuA and GpuA from P. aeruginosa to provide a structural insight into their substrate specificity. Although GbuA and GpuA share a common structural fold of the typical ureohydrolase superfamily, they exhibit significant variations in two active site loops. Mutagenesis of Met161 of GbuA and Tyr157 of GpuA, both of which are located in the active site loop 1 and predicted to be involved in substrate recognition, significantly affected their enzymatic properties, implying their important roles in catalysis.

Structural basis for the substrate recognition of peptidoglycan pentapeptides by Enterococcus faecalis VanYB.

Vancomycin resistance in Enterococci and its transfer to methicillin-resistant Staphylococcus aureus are challenging problems in health care institutions worldwide. High-level vancomycin resistance is conferred by acquiring either transposable elements of the VanA or VanB type. Enterococcus faecalis VanYB in the VanB-type operon is a d,d-carboxypeptidase that recognizes the peptidyl-d-Ala4-d-Ala5 extremity of peptidoglycan and hydrolyses the terminal d-Ala on the extracellular side of the cell wall, thereby increasing the level of glycopeptide antibiotics resistance. However, at the molecular level, it remains unclear how VanYB manipulates peptidoglycan peptides for vancomycin resistance. In this study, we have determined the crystal structures of E. faecalis VanYB in the d-Ala-d-Ala-bound, d-Ala-bound, and -unbound states. The interactions between VanYB and d-Ala-d-Ala observed in the crystal provide the molecular basis for the recognition of peptidoglycan substrates by VanYB. Moreover, comparisons with the related VanX and VanXY enzymes reveal distinct structural features of E. faecalis VanYB around the active-site cleft, thus shedding light on its unique substrate specificity. Our results could serve as the foundation for unravelling the molecular mechanism of vancomycin resistance and for developing novel antibiotics against the vancomycin-resistant Enterococcus species.

Structural Basis of the Heterodimer Formation between Cell Shape-Determining Proteins Csd1 and Csd2 from Helicobacter pylori.

Colonization of the human gastric mucosa by Helicobacter pylori requires its high motility, which depends on the helical cell shape. In H. pylori, several genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5, and csd6) play key roles in determining the cell shape by alteration of cross-linking or by trimming of peptidoglycan stem peptides. H. pylori Csd1, Csd2, and Csd3/HdpA are M23B metallopeptidase family members and may act as d,d-endopeptidases to cleave the d-Ala4-mDAP3 peptide bond of cross-linked dimer muropeptides. Csd3 functions also as the d,d-carboxypeptidase to cleave the d-Ala4-d-Ala5 bond of the muramyl pentapeptide. To provide a basis for understanding molecular functions of Csd1 and Csd2, we have carried out their structural characterizations. We have discovered that (i) Csd2 exists in monomer-dimer equilibrium and (ii) Csd1 and Csd2 form a heterodimer. We have determined crystal structures of the Csd2121-308 homodimer and the heterodimer between Csd1125-312 and Csd2121-308. Overall structures of Csd1125-312 and Csd2121-308 monomers are similar to each other, consisting of a helical domain and a LytM domain. The helical domains of both Csd1 and Csd2 play a key role in the formation of homodimers or heterodimers. The Csd1 LytM domain contains a catalytic site with a Zn2+ ion, which is coordinated by three conserved ligands and two water molecules, whereas the Csd2 LytM domain has incomplete metal ligands and no metal ion is bound. Structural knowledge of these proteins sheds light on the events that regulate the cell wall in H. pylori.

Structural and functional characterization of Helicobacter pylori DsbG.

Dsb proteins play important roles in bacterial pathogenicity. To better understand the role of Dsb proteins in Helicobacter pylori, we have structurally and functionally characterized H. pylori DsbG (HP0231). The monomer consists of two domains connected by a helical linker. Two monomers associate to form a V-shaped dimer. The monomeric and dimeric structures of H. pylori DsbG show significant differences compared to Escherichia coli DsbG. Two polyethylene glycol molecules are bound in the cleft of the V-shaped dimer, suggesting a possible role as a chaperone. Furthermore, we show that H. pylori DsbG functions as a reductase against HP0518, a putative L,D-transpeptidase with a catalytic cysteine residue.