Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris

Cyclic di-GMP is a second messenger with a role in regulation of a range of cellular functions in diverse bacteria including the virulence of pathogens. Cellular levels of cyclic di-GMP are controlled through synthesis, catalysed by the GGDEF protein domain, and degradation by EAL or HD-GYP domains. Here we report a comprehensive study of cyclic di-GMP signalling in bacterial disease in which we examine the contribution of all proteins with GGDEF, EAL or HD-GYP domains to virulence and virulence factor production in the phytopathogen Xanthomonas campestris pathovar campestris (Xcc). Genes with significant roles in virulence to plants included those encoding proteins whose probable function is in cyclic-di-GMP synthesis as well as others (including the HD-GYP domain regulator RpfG) implicated in cyclic di-GMP degradation. Furthermore, RpfG controlled expression of a subset of these genes. A partially overlapping set of elements controlled the production of virulence factors in vitro. Other GGDEF-EAL domain proteins had no effect on virulence factor synthesis but did influence motility. These findings indicate the existence of a regulatory network that may allow Xcc to integrate information from diverse environmental inputs to modulate virulence factor synthesis as well as of cyclic di-GMP signalling systems dedicated to other specific tasks.     

The HD-GYP domain, cyclic di-GMP signaling, and bacterial virulence to plants

Cyclic di-GMP is an almost ubiquitous second messenger in bacteria that was first described as an allosteric activator of cellulose synthase but is now known to regulate a range of functions, including virulence in human and animal pathogens. Two protein domains, GGDEF and EAL, are implicated in the synthesis and degradation, respectively, of cyclic di-GMP. These domains are widely distributed in bacteria, including plant pathogens. The majority of proteins with GGDEF and EAL domains contain additional signal input domains, suggesting that their activities are responsive to environmental cues. Recent studies have demonstrated that a third domain, HD-GYP, is also active in cyclic di-GMP degradation. In the plant pathogen Xanthomonas campestris pv. campestris, a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC positively controls virulence. The signals recognized by RpfC may include the cell-cell signal DSF, which also acts to regulate virulence in X. campestris pv. campestris. Here, we review these recent advances in our understanding of cyclic di-GMP signaling with particular reference to one or more roles in the bacterial pathogenesis of plants.     

Structural Insight into the Mechanism of c-di-GMP Hydrolysis by EAL Domain Phosphodiesterases

Cyclic diguanylate (or bis-(3′-5′) cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 Å) and in complex with c-di-GMP (2.35 Å), and unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.     

The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains

The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg(2+) or Mn(2+), strongly inhibited by Ca(2+), and independent of protein oligomerization. Linear dimeric GMP was shown to be 5'pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5'pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.     

The Functional Role of a Conserved Loop in EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterase

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/α)₈ barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K_m) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K_m correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg²⁺ ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.The cyclic dinucleotide cyclic di-GMP (c-di-GMP) has emerged as a major bacterial messenger for mediating a variety of cellular functions that range from virulence expression and biofilm formation (5, 14, 30). The cellular concentration of c-di-GMP is controlled by the GGDEF domain proteins with diguanylate cyclase (DGC) activity and the EAL domain proteins with c-di-GMP-specific phosphodiesterase (PDE) activity. GGDEF domains catalyze the synthesis of c-di-GMP from GTP, whereas EAL domains catalyze the hydrolysis of c-di-GMP to generate the linear 5′-pGpG. Although a family of HD-GYP domain proteins has also been found as c-di-GMP-specific PDEs, the overwhelmingly large number of genes encoding the EAL domains in bacterial genomes suggests that the EAL domains are the major PDEs for maintaining the cellular c-di-GMP concentration. Remarkably, multiple copies of EAL domain-encoding genes are usually found in bacterial cells, with as many as 21 in Pseudomonas aeruginosa and 32 in Vibrio cholerae. Although many of the EAL domains were found to function as PDE domains for c-di-GMP degradation, emerging evidence suggests that some EAL domains function as ligand- or protein-binding domains without catalytic activity (24, 28, 40).The detailed structure and catalytic mechanism of the EAL domains have started to be elucidated recently. The crystal structures of two proteins with EAL domains, TdEAL and YkuI, have been determined (Protein Data Bank accession nos. 2BAS, 2R6O, and 2w27) (23). EAL domains adopt a (β/α)₈ barrel fold that contains two extended strands, including an antiparallel strand. The (β/α)₈ barrel fold, first found in triosephosphate isomerase, has been observed in a diversity of enzymes that include many hydrolyases and isomerases (34). Similar to other (β/α)₈ barrel fold enzymes, the catalytic residues of the EAL domain are located at the C-terminal ends of the β-strands and the beginning of the β→α loops connecting the β-strands and α-helices. In the proposed mechanism, EAL domains catalyze the hydrolysis of c-di-GMP by using a Mg²⁺ ion and a general base catalyst (Glu) for generating the nucleophilic H₂O (28). The catalytic mechanism is supported by the crystal structure of the YkuI-substrate binary complex (Protein Data Bank accession no. 2w27) and the model of the TdEAL-substrate complex (23, 28). Both structures showed that the EAL domains bind c-di-GMP in such a configuration that the scissile phosphorus-oxygen bond aligns linearly with the attaching water and the general base catalyst. The catalytic mechanism can account for the lack of catalytic activity for most known inactive EAL domains, with the loss of enzymatic activity arising from the absence of the general base catalyst and/or the residues that coordinate the Mg²⁺ ion (28).It is well-known that many (β/α)₈ barrel fold enzymes contain a flexible active site loop between the β6 strand and α6 helix (34). Despite the diverse reactions catalyzed by (β/α)₈ barrel fold enzymes, this extended loop, often referred to as loop 6, plays an important role as a functional lid for substrate sequestering, solvent exclusion, and product release (15). The loop was found to facilitate substrate binding and conformational transition in tryptophan synthase (3, 4) and functions as a lid for substrate sequestering during catalysis in inosine 5′-monophosphate dehydrogenase (22). Notably, it was shown that the loop sways from the active site in the nonactive structure of ribulose-1,5-bisphosphate carboxylase but folds over to shield the active site from the solvent in the activated structure (21). Similar functions have also been proposed for loop 6 in other (β/α)₈ barrel fold enzymes, such as triosephosphate isomerase and phosphoriboxyl anthranilate isomerase (15, 25, 26). Hence, it seems that the functional role of loop 6 has been well preserved in (β/α)₈ barrel fold enzymes during evolution. The (β/α)₈ barrel folded EAL domains also contain an eight-residue loop between the β6 strand and α6 helix that seems to be critical for catalysis. Schmidt and coworkers (32) first noticed that the catalytically active EAL domains seem to contain a conserved motif that was later confirmed to contain loop 6 [DFG(T/A)GYSS] and one of the residues (Asp) for Mg²⁺ binding (28, 32). We previously noticed that mutation of the essential catalytic residues is usually accompanied by the degeneration of loop 6 in catalytically inactive EAL domains (28). Moreover, we observed that the mutation of a residue interacting with loop 6 in the EAL domain-containing RocR abolished enzymatic activity, which led us to postulate a critical role for loop 6 in catalysis (28).To elucidate the precise role played by loop 6 in c-di-GMP hydrolysis, we examined three EAL domain-containing proteins that include RocR, PA2567, and A. xylinus DGC2. The residues of loop 6 [DFG(A/T)SYSS] in RocR and PA2567 are well conserved, as observed in other catalytically active EAL domains. We show that mutations in the loop 6 region in RocR and PA2567 had significant effect on the structure and catalysis of the EAL domain. By using the method of hydrogen-deuterium (H/D) exchange-coupled mass spectrometry, we demonstrated that a single remote mutation in the phosphoreceiver domain of RocR caused correlated changes in loop 6 conformation and catalytic properties. We further show that the catalytic activity of the inactive EAL domain of A. xylinus DGC2 can be recovered by restoring loop 6. The functional roles of loop 6 in EAL domains in substrate binding and catalysis were discussed in conjunction with the structural data for two EAL domains.     

Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.     

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg²⁺, cdG·Ca²⁺ complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.     

水稻白叶枯病菌EAL结构域蛋白VieAxoo基因缺失突变和功能分析

摘要：【目的】旨在揭示水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, 简称Xoo) 环鸟苷二磷酸（c-di-GMP）信号蛋白VieAxoo的生物学功能。【方法】本研究通过标记置换法对vieAxoo基因(PXO_04753)进行了缺失突变研究,采用表性测定进行了部分功能鉴定。【结果】从野生型菌株PXO99A中克隆的vieAxoo基因序列与其它病原黄单胞菌的同源序列高度保守。VieAxoo具有参与c-di-GMP降解的磷酸二酯酶（PDE）EAL结构域和磷酸信号识别受体REC结构域     

Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage

A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex. The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD. We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis. It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations. The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue. A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.     

The 1.3-Angstrom-resolution crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae

The beta-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 A resolution crystal structure of the beta-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated betaalphabetaalphabetaalphabetabeta fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg(2+) ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.     

Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthetase are involved in tRNA and Tyr-AMP binding

Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.     

Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus

The solution structure of the mouse pro-hormone convertase (PC) 1 pro-domain was determined using heteronuclear NMR spectroscopy and is the first structure to be obtained for any of the domains in the convertase family. The ensemble of NMR-derived structures shows a well-ordered core consisting of a four-stranded antiparallel beta-sheet with two alpha-helices packed against one side of this sheet. Sequence homology suggests that the other eukaryotic PC pro-domains will have the same overall fold and most of the residues forming the hydrophobic core of PC1 are highly conserved within the PC family. However, some of the core residues are predicted by homology to be replaced by polar amino acid residues in other PC pro-domains and this may help to explain their marginal stability. Interestingly, the folding topology observed here is also seen for the pro-domain of bacterial subtilisin despite little or no sequence homology. Both the prokaryotic and eukaryotic structures have hydrophobic residues clustered on the solvent-accessible surface of their beta-sheets although the individual residue types differ. In the bacterial case this region is buried at the binding interface with the catalytic domain and, in the eukaryotic PC family, these surface residues are conserved. We therefore propose that the hydrophobic patch in the PC1 pro-domain is involved in the binding interface with its cognate catalytic domain in a similar manner to that seen for the bacterial system. The PC1 pro-domain structure also reveals potential mechanisms for the acid-induced dissociation of the complex between pro- and catalytic domains.     

Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage

Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.     

Functional analysis of ISC1 by site-directed mutagenesis

We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p [Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798]. The present study was conducted to determine specific domains in Isc1p required for catalysis. Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1. Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues. Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins. In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain". G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis. D163A and K168A lost activity completely. Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity. The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p. Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding. Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain. This study provides novel insight into a new domain found in Isc1p and related enzymes.     

Determination of metal ion binding sites within the hairpin ribozyme domains by NMR

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains, A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn(2+), Mg(2+), and Co(NH(3))(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH(3))(6)(3+) or Mg(2+). No specific monovalent ion binding sites exist on the B domain, as determined by (15)NH(4)(+) binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn(2+) interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H(2)O)(6)(2+) or Co(NH(3))(6)(3+). Site 1 is very similar in geometry to a site within the P4-P6 domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.     

Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the γ-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg(2+) for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.     

Essential roles of innersphere metal ions for the formation of the glutamine binding site in a bifunctional ribozyme.

Numerous studies on naturally occurring ribozymes have shown that the functional roles of metal ions in promoting RNA catalysis are diverse. Earlier studies performed on the in vitro selected aminoacyl-transferase ribozyme (ATRib) have revealed that a fully hydrated Mg2+ ion plays an essential role in catalysis [Suga, H., Cowan, J. A., and Szostak, J. W. (1998) Biochemistry 28, 10118-10125]. More recently, we have evolved this ATRib into a bifunctional ribozyme, called AD02 [Lee, N., et al. (2000) Nat. Struct. Biol. 7, 28-33]. This new ribozyme consists of two catalytic domains, the original ATRib domain and a new glutamine-recognition (QR) domain, and exhibits a function of charging glutamine to tRNA. Here we elucidate crucial roles of metal ions involved in the QR domain, that are distinct from those in the ATRib domain. The metal ions in the QR domain require innersphere coordinations, and both Mg2+ and Ca2+ can support catalysis. Extensive Tb3+-Mg2+ and Tb3+-Co(NH3)6(3+) competition cleavage experiments have shown that the QR domain has high and low affinity metal binding sites, which are involved in the Mg2+-dependent structural alteration to form the glutamine binding site [Lee, N., and Suga, H. (2001) RNA 7, 1043-1051]. Kinetic studies in the presence of divalent and monovalent ions have suggested that the essential role of the metal ions in the QR domain is most likely structural.     

The catalytic domain of endoglucanase A from Clostridium cellulolyticum: effects of arginine 79 and histidine 122 mutations on catalysis.

Sequence analysis of the endoglucanase EGCCA of Clostridium cellulolyticum indicates the existence of two domains: a catalytic domain extending from residue 1 to residue 376 and a reiterated domain running from residue 390 to 450. A small deletion in the C terminal end of the catalytic domain inactivated the protein. From the analysis of the sequences of 26 endoglucanases belonging to family A, we focused on seven amino acids which were totally conserved in all the catalytic domains compared. The roles of two of these, Arg-79 and His-122, were studied and defined on the basis of the mutants obtained by introducing various substitutions. Our findings suggest that Arg-79 is involved in the structural organization of the protein; the His-122 residue seems to be more essential for catalysis. The role of His-123, which is conserved only in subfamily A4, was also investigated.     

Structural insights into single-stranded DNA binding and cleavage by F factor TraI

Conjugative plasmid transfer between bacteria disseminates antibiotic resistance and diversifies prokaryotic genomes. Relaxases, proteins essential for conjugation, cleave one plasmid strand sequence specifically prior to transfer. Cleavage occurs through a Mg(2+)-dependent transesterification involving a tyrosyl hydroxyl and a DNA phosphate. The structure of the F plasmid TraI relaxase domain, described here, is a five-strand beta sheet flanked by alpha helices. The protein resembles replication initiator protein AAV-5 Rep but is circularly permuted, yielding a different topology. The beta sheet forms a binding cleft lined with neutral, nonaromatic residues, unlike most single-stranded DNA binding proteins which use aromatic and charged residues. The cleft contains depressions, suggesting base recognition occurs in a knob-into-hole fashion. Unlike most nucleases, three histidines but no acidic residues coordinate a Mg(2+) located near the catalytic tyrosine. The full positive charge on the Mg(2+) and the architecture of the active site suggest multiple roles for Mg(2+) in DNA cleavage.     

Modeling a dehalogenase fold into the 8-A density map for Ca(2+)-ATPase defines a new domain structure

Members of the large family of P-type pumps use active transport to maintain gradients of a wide variety of cations across cellular membranes. Recent structures of two P-type pumps at 8-A resolution have revealed the arrangement of transmembrane helices but were insufficient to reveal the architecture of the cytoplasmic domains. However, recent proposals of a structural homology with a superfamily of hydrolases offer a new basis for modeling these domains. In the current work, we have extended the sequence comparison for the superfamily and delineated domains in the 8-A density map of Ca(2+)-ATPase. The homology suggests a new domain structure for Ca(2+)-ATPase and, specifically, that the phosphorylation domain adopts a Rossman fold. Accordingly, the atomic structure of L-2 haloacid dehalogenase has been fitted into the relevant domain of Ca(2+)-ATPase. The resulting model suggests the existence of two ATP sites at the interface between two domains. Based on this new model, we are able to reconcile numerous results of mutagenesis and chemical cross-linking within the catalytic domains. Furthermore, we have used the model to predict the configuration of Mg.ATP at its binding site. Based on this prediction, we propose a mechanism, involving a change in Mg(2+) liganding, for initiating the domain movements that couple sites of ion transport to ATP hydrolysis.