Takato Yano

Crystal Structure of the Peptidase Domain of Streptococcus ComA, a Bifunctional ATP-binding Cassette Transporter Involved in the Quorum-sensing Pathway

ComA of Streptococcus is a member of the bacteriocin-associated ATP-binding cassette transporter family and is postulated to be responsible for both the processing of the propeptide ComC and secretion of the mature quorum-sensing signal. The 150-amino acid peptidase domain (PEP) of ComA specifically recognizes an extended region of ComC that is 15 amino acids in length. It has been proposed that an amphipathic α-helix formed by the N-terminal leader region of ComC, as well as the Gly-Gly motif at the cleavage site, is critical for the PEP-ComC interaction. To elucidate the substrate recognition mechanism, we determined the three-dimensional crystal structure of Streptococcus mutans PEP and then constructed models for the PEP·ComC complexes. PEP had an overall structure similar to the papain-like cysteine proteases as has long been predicted. The active site was located at the bottom of a narrow cleft, which is suitable for binding the Gly-Gly motif. Together with the results from mutational experiments, a shallow hydrophobic concave surface of PEP was proposed as a site that accommodates the N-terminal helix of ComC. This dual mode of substrate recognition would provide the small PEP domain with an extremely high substrate specificity.

Expression and Characterization of the Peptidase Domain of Streptococcus pneumoniae ComA, a Bifunctional ATP-binding Cassette Transporter Involved in Quorum Sensing Pathway

ComA, a member of the bacteriocin ATP-binding cassette transporters, is postulated to be responsible for both the processing of the propeptide ComC and secretion of the mature competence-stimulating peptide, which regulates the competence and subsequent genetic transformation in Streptococcus pneumoniae. A recombinant N-terminal peptidase domain of ComA, designated PEP, was expressed as a soluble protein in Escherichia coli, purified to homogeneity, and characterized. Gel-filtration analysis revealed that PEP functions as a monomer. The purified PEP exhibited an efficient proteolytic activity for the substrate ComC, which was cleaved after the double glycine site. The stability of PEP was examined by circular dichroism analyses. A convenient method for analyzing the proteolytic activity of PEP was developed, and the kinetic parameters for ComC were determined (kcat = 1.5 ± 0.083 min–1 and Km = 62 ± 9.0 μm). Replacements of Cys17 of PEP with Ser or Ala and His96 with Ala resulted in complete loss of activity, indicating that both Cys17 and His96 are essential for the catalysis. Together with information from a protease data base, the N-terminal domain of ComA was concluded to belong to the same clan as the papain-like cysteine proteases. Mutant substrates, in which each of the double glycines was replaced with Ala, were cleaved very poorly by PEP. The mechanism of this strict substrate specificity is discussed on the basis of the sequence alignment with other cysteine proteases.

Substrate recognition mechanism of the peptidase domain of the quorum-sensing-signal-producing ABC transporter ComA from Streptococcus.

ComA of Streptococcus is a member of the bacteriocin-associated ABC transporters, which is responsible for both the processing of the propeptide ComC and secretion of the mature quorum-sensing signal. The quorum-sensing system is a bacterial intercellular communication system implicated in various functions including biofilm formation. In this study, the peptidase domains (PEPs) of the ComAs from six species of Streptococcus and ComCs from four species were expressed, purified, and characterized to address the mechanism of the substrate recognition of PEP. PEPs specifically cleaved ComCs after the Gly-Gly site in all the PEP-ComC combinations examined. The N-terminal leader region of ComC was found to form an amphiphilic alpha-helix structure upon binding to the PEP. Furthermore, mutagenesis studies revealed that four conserved hydrophobic residues in this leader region of ComC extending from -15 to -4 positions are critical in the interaction with PEP. Together with the double glycine motif, these structural features of ComC would explain the strict substrate specificity of the PEP.

ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex

Significance The cathelicidin antimicrobial peptide (CAMP) is an innate immune element that promotes antimicrobial defense, but excessive CAMP can stimulate inflammation and tumorigenesis. We recently discovered that external perturbations that induce subtoxic levels of endoplasmic reticulum (ER) stress increase sphingosine-1-phosphate (S1P) production, in turn activating NF-κB–mediated CAMP synthesis. We report here that S1P interacts with the heat shock proteins (HSP90α and GRP94) through a previously unidentified S1P receptor-independent intracellular mechanism, followed by the activation of NF-κB leading to stimulation of CAMP production. These studies illuminate the critical role of both ER stress and S1P in orchestrating stress-specific signals that enhance innate immunity. We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

Sulfur Modifications of the Wobble U34 in tRNAs and their Intracellular Localization in Eukaryotic Cells

The wobble uridine (U34) of transfer RNAs (tRNAs) for two-box codon recognition, i.e., tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG, harbor a sulfur- (thio-) and a methyl-derivative structure at the second and fifth positions of U34, respectively. Both modifications are necessary to construct the proper anticodon loop structure and to enable them to exert their functions in translation. Thio-modification of U34 (s2U34) is found in both cytosolic tRNAs (cy-tRNAs) and mitochondrial tRNAs (mt-tRNAs). Although l-cysteine desulfurase is required in both cases, subsequent sulfur transfer pathways to cy-tRNAs and mt-tRNAs are different due to their distinct intracellular locations. The s2U34 formation in cy-tRNAs involves a sulfur delivery system required for the biosynthesis of iron-sulfur (Fe/S) clusters and certain resultant Fe/S proteins. This review addresses presumed sulfur delivery pathways for the s2U34 formation in distinct intracellular locations, especially that for cy-tRNAs in comparison with that for mt-tRNAs.

Proteome-wide identification of lysine succinylation in thermophilic and mesophilic bacteria

Lysine succinylation, one of post-translational acylations conserved from eukaryotes to bacteria, plays regulatory roles in various cellular processes. However, much remains unknown about the general and specific characteristics of lysine succinylation among bacteria, and about its functions different from those of other acylations. In this study, we characterized lysine succinylation, a newly discovered widespread type of lysine acylation in five bacterial species with different characteristics such as optimal growth temperature and cell wall structure. This study is the first to demonstrate that succinylation is general phenomenon occurring not only in mesophiles but also in thermophiles. Mapping of succinylation sites on protein structures revealed that succinylation occurs at many lysine residues important for protein function. Comparison of the succinylation sites in the five bacterial species provides insights regarding common protein regulation mechanisms utilizing lysine succinylation. Many succinylation sites were conserved among five bacteria, especially between Geobacillus kaustophilus and Bacillus subtilis, some of which are functionally important sites. Furthermore, systematic comparison of the succinyl-proteome results and our previous propionyl-proteome results showed that the abundance of these two types of acylations is considerably different among the five bacteria investigated. Many succinylation and propionylation events were detected in G. kaustophilus, whereas Escherichia coli and B. subtilis exhibited high succinylation and low propionylation; low succinylation and high propionylation were identified in Thermus thermophilus, and low succinylation and propionylation were observed in Rhodothermus marinus. Comparison of the characteristics of lysine succinylation and lysine propionylation suggested these two types of acylation play different roles in cellular processes.

Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL

In early reactions of DNA mismatch repair, MutS recognizes mismatched bases and activates MutL endonuclease to incise the error-containing strand of the duplex. DNA sliding clamp is responsible for directing the MutL-dependent nicking to the newly synthesized/error-containing strand. In Bacillus subtilis MutL, the β-clamp-interacting motif (β motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with β-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until β clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the β motif. For example, the region corresponding to the β motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the β motif are not conserved in Thermus thermophilus MutL. We then revealed the 1.35 Å-resolution crystal structure of A. aeolicus MutL CTD, which lacks the β motif but retains the metal-binding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of β motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of β-clamp and that β-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a β motif-containing MutL, required β-clamp for the digestion of DNA. We propose that MutL endonucleases are divided into three subfamilies on the basis of their structural features and dependence on β-clamp.

Boundary of the nucleotide-binding domain of Streptococcus ComA based on functional and structural analysis.

The ATP-binding cassette (ABC) transporter ComA is a key molecule essential for the first step of the quorum-sensing system of Streptococcus. The nucleotide binding domains (NBD) of Streptococcus mutans ComA with different N termini, NBD1 (amino acid residues 495-760), NBD2 (517-760), and NBD3 (528-760), were expressed, purified, and characterized. The shortest NBD3 corresponds to the region commonly defined as NBD in the database searches of ABC transporters. A kinetic analysis showed that the extra N-terminal region conferred a significantly higher ATP hydrolytic activity on the NBD at a neutral pH. Gel-filtration, X-ray crystallography, and mutational analyses suggest that at least four to five residues beyond the N-terminal boundary of NBD3 indeed participate in stabilizing the protein scaffold of the domain structure, thereby facilitating the ATP-dependent dimerization of NBD which is a prerequisite to the catalysis. These findings, together with the presence of a highly conserved glycine residue in this region, support the redefinition of the N-terminal boundary of the NBD of these types of ABC exporters.

Indispensable residue for uridine binding in the uridine-cytidine kinase family

Uridine-cytidine kinase (UCK), including human UCK2, are a family of enzymes that generally phosphorylate both uridine and cytidine. However, UCK of Thermus thermophilus HB8 (ttCK) phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on Tyr93, although the precise mechanism remains unresolved. Exhaustive mutagenesis of Tyr93 in ttCK revealed that the uridine phosphorylation activity was restored only by replacement of Tyr93 with His or Gln. Replacement of His117 in human UCK2, corresponding to residue Tyr93 in ttCK, by Tyr resulted in a loss of uridine phosphorylation activity. These findings indicated that uridine phosphorylation activity commonly depends on a single residue in the UCK family.

The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair

Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. RecA/Rad51 paralogs have been recognized as an important factor of HR. Among them, only one bacterial RecA/Rad51 paralog, RadA, is involved in HR as an accessory factor of RecA recombinase. RadA has a unique Lon protease-like domain (LonC) at its C terminus, in addition to a RecA-like ATPase domain. Unlike Lon protease, RadAs LonC domain does not show protease activity but is still essential for RadA-mediated DNA repair. Reconciling these two facts has been difficult because RadAs tertiary structure and molecular function are unknown. Here, we describe the hexameric ring structure of RadAs LonC domain, as determined by X-ray crystallography. The structure revealed the two positively charged regions unique to the LonC domain of RadA are located at the intersubunit cleft and the central hole of a hexameric ring. Surprisingly, a functional domain analysis demonstrated the LonC domain of RadA binds DNA, with site-directed mutagenesis showing that the two positively charged regions are critical for this DNA-binding activity. Interestingly, only the intersubunit cleft was required for the DNA-dependent stimulation of ATPase activity of RadA, and at least the central hole was essential for DNA repair function. Our data provide the structural and functional features of the LonC domain and their function in RadA-mediated DNA repair.