Characterization of Elements Involved in Allosteric Light Regulation of Phosphodiesterase Activity by Comparison of Different Functional BlrP1 States

Bacteria have evolved dedicated signaling mechanisms that enable the integration of a range of environmental stimuli and the accordant modulation of metabolic pathways. One central signaling molecule in bacteria is the second messenger cyclic dimeric GMP (c-di-GMP). Complex regulatory mechanisms for modulating c-di-GMP concentrations have evolved, in line with its importance for maintaining bacterial fitness under changing environmental conditions. One interesting example in this context is the blue-light-regulated phosphodiesterase 1 (BlrP1) of Klebsiella pneumoniae. This covalently linked system of a sensor of blue light using FAD (BLUF) and an EAL phosphodiesterase domain orchestrates the light-dependent down-regulation of c-di-GMP levels. To reveal details of light-induced structural changes involved in EAL activity regulation, we extended previous crystallographic studies with hydrogen–deuterium exchange experiments and small-angle X-ray scattering analysis of different functional BlrP1 states. The combination of hydrogen–deuterium exchange and small-angle X-ray scattering allows the integration of local and global structural changes and provides an improved understanding of light signaling via an allosteric communication pathway between the BLUF and EAL domains. This model is supported by results from a mutational analysis of the EAL dimerization region and the analysis of metal-coordination effects of the EAL active site on the dark-state recovery kinetics of the BLUF domain. In combination with structural information from other EAL domains, the observed bidirectional communication points to a general mechanism of EAL activity regulation and suggests that a similar allosteric coupling is maintained in catalytically inactive EAL domains that retain a regulatory function.     

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.     

Transmembrane redox control and proteolysis of PdeC,a novel type of c‐di‐GMP phosphodiesterase

The nucleotide second messenger c‐di‐GMP nearly ubiquitously promotes bacterial biofilm formation, with enzymes that synthesize and degrade c‐di‐GMP being controlled by diverse N‐terminal sensor domains. Here, we describe a novel class of widely occurring c‐di‐GMP phosphodiesterases (PDE) that feature a periplasmic “CSS domain” with two highly conserved cysteines that is flanked by two transmembrane regions (TM1 and TM2) and followed by a cytoplasmic EAL domain with PDE activity. Using PdeC, one of the five CSS domain PDEs of Escherichia coli K‐12, we show that DsbA/DsbB‐promoted disulfide bond formation in the CSS domain reduces PDE activity. By contrast, the free thiol form is enzymatically highly active, with the TM2 region promoting dimerization. Moreover, this form is processed by periplasmic proteases DegP and DegQ, yielding a highly active TM2 + EAL fragment that is slowly removed by further proteolysis. Similar redox control and proteolysis was also observed for a second CSS domain PDE, PdeB. At the physiological level, CSS domain PDEs modulate production and supracellular architecture of extracellular matrix polymers in the deeper layers of mature E. coli biofilms.     

Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization

Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca²⁺-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca²⁺ signaling since Ca²⁺- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.     

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.     

Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa

EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second-messenger cyclic di-GMP in bacteria. Given the pivotal roles of EAL domains in the regulation of many bacterial behaviors, the elucidation of their catalytic and regulatory mechanisms would contribute to the effort of deciphering the cyclic di-GMP signaling network. Here, we present data to show that RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO-1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg(2+) ion. In addition to the five essential residues involved in Mg(2+) binding, we propose that the essential residue E(352) functions as a general base catalyst assisting the deprotonation of Mg(2+)-coordinated water to generate the nucleophilic hydroxide ion. The mutation of other conserved residues caused various degree of changes in the k(cat) or K(m), leading us to propose their roles in residue positioning and substrate binding. With functions assigned to the conserved groups in the active site, we discuss the molecular basis for the lack of activity of some characterized EAL domain proteins and the possibility of predicting the phosphodiesterase activities for the vast number of EAL domains in bacterial genomes in light of the catalytic mechanism.     

Chemical shift assignments of the connexin45 carboxyl terminal domain: monomer and dimer conformations

Connexin45 (Cx45) is a gap junction protein involved in cell-to-cell communication in the heart and other tissues. Here we report the ¹H, ¹⁵N, and ¹³C resonance assignments for the monomer and dimer conformations of the Cx45 carboxyl terminal (Cx45CT) domain and provide evidence of dimerization using diffusion ordered spectroscopy. The predicted secondary structure of the Cx45CT domain based on the chemical shifts identified one region of α-helical structure, which corresponds to the residues that broadened beyond detection in the dimer confirmation. Previous biophysical studies from our laboratory characterizing the CT domain from the other major cardiac connexins, Cx40 and Cx43, suggest that the amount of α-helical content may translate into the ability of a protein to dimerize. Even though the CT domain is thought to be the main regulatory domain of most connexins, the physiological role of CT dimerization is currently unknown. Therefore, these assignments will be useful for determining the intermolecular interactions that mediate Cx45CT dimerization, information that will be used to characterize dimerization in functional channels, as well as characterizing the binding sites for molecular partners involved in Cx45 regulation.     

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg²⁺ deprivation, but previous work suggested that MgtC is not a Mg²⁺ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg²⁺ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg²⁺ concentration, indicating that it does not bind Mg²⁺. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg²⁺ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.     

Crystal Structures of YkuI and Its Complex with Second Messenger Cyclic Di-GMP Suggest Catalytic Mechanism of Phosphodiester Bond Cleavage by EAL Domains

Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca²⁺. The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel β-strand. The complex with c-di-GMP-Ca²⁺ defines the active site of the putative phosphodiesterase located at the C-terminal end of the β-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PAS-like domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed.The dinucleotide cyclic di-GMP (c-di-GMP) was discovered about 20 years ago when it was found to regulate the activity of cellulase synthase in Acetobacter xylinum (1). However, its prominent role as a global second messenger has been realized only upon the recent recognition of the omnipresence of genes coding for domains that catalyze c-di-GMP biosynthesis and degradation in eubacteria (2). GGDEF domains catalyze the condensation of two GTP molecules to the cyclic 2-fold symmetric dinucleotide (diguanylate cyclase activity (3-6)), whereas EAL domains are involved in its degradation to yield the linear dinucleotide pGpG (phosphodiesterase (PDE)⁴ A activity) (3, 7-9). Recently, also HD-GYP domains have been implicated in c-di-GMP-specific PDE activity (10). All the domains have been named according to their sequence signature motifs. They are typically found in combinations with various other, mostly sensory or regulatory, domains. It is thought that the balance between antagonistic diguanylate cyclase and PDE-A activities determines the cellular level of c-di-GMP and, thus, affects a variety of physiological processes in bacteria.It has been shown that, in general, c-di-GMP regulates cell surface-associated traits and community behavior such as biofilm formation (for reviews see Refs. 11-12), and its relevance to the virulence of pathogenic bacteria has been demonstrated (11, 13, 14). In particular, the dinucleotide has been proposed to orchestrate the switch between acute and persistent phase of infection.The best characterized diguanylate cyclase is PleD from Caulobacter crescentus with a Rec-Rec-GGDEF domain architecture (Rec indicates response regulator receiver domain). The structure of its GGDEF domain revealed a single GTP-binding site and suggested that dimerization is the prerequisite for enzymatic activity (4). This has been corroborated recently by crystallography showing directly that modification of the first Rec domain, mimicking phosphorylation by the cognate kinase, induces formation of a tightly packed dimer (15). Additionally, an upper limit of c-di-GMP levels in the cell seems to be ensured by potent allosteric product inhibition of the PleD cyclase (4, 15, 16). Recently, the crystal structure of another diguanylate cyclase, WspR from Pseudomonas aeruginosa with a Rec-GGDEF domain architecture, has been determined (17), which showed a tetrameric quaternary structure and active and feedback inhibition sites that are very similar to those in PleD.For EAL domains, it has been demonstrated that genetic knock-out results in phenotypes that are in line with the paradigm that an elevated cellular c-di-GMP concentration corresponds to a sessile and a low concentration to a motile bacterial life style (13, 18, 19). Only recently, EAL-mediated PDE-A activity has been measured in vitro (7-9, 20-22).The Bacillus subtilis YkuI protein was targeted for structure determination by the Midwest Center for Structural Genomics as a member of the large sequence family that contains EAL (Pfam number PF00563) domains. Here we report the crystal structure of YkuI showing the fold of the N-terminal EAL domain and the C-terminal PAS-like domain. Co-crystallization with c-di-GMP revealed the substrate binding mode and allows the proposal of a catalytic mechanism. The PAS-like domain most probably has regulatory function, which is discussed. Recently, another EAL structure has been deposited in the Protein Data Bank by the Midwest Center for Structural Genomics, the EAL domain of a GGDEF-EAL protein from Thiobacillus denitrificans (tdEAL; PDB code 2r6o). Comparison of the two structures suggests a possible regulatory mechanism.     

Crystal structure of an HD‐GYP domain cyclic‐di‐GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre

Bis‐(3′,5′) cyclic di‐guanylate (c‐di‐GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c‐di‐GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD‐GYP domains. Here, we have determined the structure of an enzymatically active HD‐GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c‐di‐GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c‐di‐GMP. This structure completes the picture of all domains involved in c‐di‐GMP metabolism and reveals that the HD‐GYP family splits into two distinct subgroups containing bi‐ and trinuclear metal centres.     

Domain Structure of Virulence-associated Response Regulator PhoP of Mycobacterium tuberculosis: ROLE OF THE LINKER REGION IN REGULATOR-PROMOTER INTERACTION(S)

The PhoP and PhoR proteins from Mycobacterium tuberculosis form a highly specific two-component system that controls expression of genes involved in complex lipid biosynthesis and regulation of unknown virulence determinants. The several functions of PhoP are apportioned between a C-terminal effector domain (PhoPC) and an N-terminal receiver domain (PhoPN), phosphorylation of which regulates activation of the effector domain. Here we show that PhoPN, on its own, demonstrates PhoR-dependent phosphorylation. PhoPC, the truncated variant bearing the DNA binding domain, binds in vitro to the target site with affinity similar to that of the full-length protein. To complement the finding that residues spanning Met¹ to Arg¹³⁸ of PhoP constitute the minimal functional PhoPN, we identified Arg¹⁵⁰ as the first residue of the distal PhoPC domain capable of DNA binding on its own, thereby identifying an interdomain linker. However, coupling of two functional domains together in a single polypeptide chain is essential for phosphorylation-coupled DNA binding by PhoP. We discuss consequences of tethering of two domains on DNA binding and demonstrate that linker length and not individual residues of the newly identified linker plays a critical role in regulating interdomain interactions. Together, these results have implications for the molecular mechanism of transmission of conformation change associated with phosphorylation of PhoP that results in the altered DNA recognition by the C-terminal domain.     

Identification of the divalent metal ion binding domain of myosin regulatory light chains using spin-labelling techniques

By the criterion of their primary structure myosin regulatory light chains belong to the ‘calcium binding protein’ family and are thought to contain domains related to the E-F hand structure found in parvalbumin. However, the presence of deletions and non-conservative substitutions in the regulatory light chains indicates that, of the four domains apparent in their structure, only the first is competent to bind Ca²⁺ or other divalent metal ions. Electron paramagnetic resonance studies were performed in an attempt to provide experimental verification of this hypothesis. The approach is based on the finding that the paramagnetic Mn²⁺ ion substitutes for Ca²⁺ at the divalent metal ion site and that different regulatory light-chain isotypes contain cysteine residues in different domains which may be spin-labelled with a nitroxide derivative. The electron spin interaction between these two paramagnetic centres is a function of the distance of their separation. Clam (Mercenaria mercenaria) regulatory light chain contains a single cysteine residue located near the first domain and, when spin-labelled, the intensity of the nitroxide signal is reduced by 25% on binding one mole of Mn²⁺. Rabbit skeletal regulatory light chain contains two cysteine residues located in the third and fourth domains and no (<5%) interaction is observed when Mn²⁺ binds to spin-labelled derivatives. Qualitatively, these results suggest that domain 1 is the most likely candidate for the Mn²⁺ binding site. A more quantitative evaluation using the Leigh (1970) theory for the dipolar coupling between rigid-lattice electron spins and various models for the regulatory light chain tertiary structure, including that predicted by Kretsinger &; Barry (1975) for the possibly isologous troponin C structure, substantiates this conclusion.     

Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae

Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.     

The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ

The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of phosphatase activity and Ca²⁺ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.     

Structural distinctions between NAD+ riboswitch domains 1 and 2 determine differential folding and ligand binding

Riboswitches are important gene regulatory elements frequently encountered in bacterial mRNAs. The recently discovered nadA riboswitch contains two similar, tandemly arrayed aptamer domains, with the first domain possessing high affinity for nicotinamide adenine dinucleotide (NAD⁺). The second domain which comprises the ribosomal binding site in a putative regulatory helix, however, has withdrawn from detection of ligand-induced structural modulation thus far, and therefore, the identity of the cognate ligand and the regulation mechanism have remained unclear. Here, we report crystal structures of both riboswitch domains, each bound to NAD⁺. Furthermore, we demonstrate that ligand binding to domain 2 requires significantly higher concentrations of NAD⁺ (or ADP retaining analogs) compared to domain 1. Using a fluorescence spectroscopic approach, we further shed light on the structural features which are responsible for the different ligand affinities, and describe the Mg²⁺-dependent, distinct folding and pre-organization of their binding pockets. Finally, we speculate about possible scenarios for nadA RNA gene regulation as a putative two-concentration sensor module for a time-controlled signal that is primed and stalled by the gene regulation machinery at low ligand concentrations (domain 1), and finally triggers repression of translation as soon as high ligand concentrations are reached in the cell (domain 2).     

Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by ‘donor-strand complementation.’ Furthermore, we identified a conserved metal (Ni²⁺) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly.     

Novel Mechanism for Cyclic Dinucleotide Degradation Revealed by Structural Studies of Vibrio Phosphodiesterase V-cGAP3

3′3′-cyclic GMP–AMP (3′3′-cGAMP) belongs to a family of the bacterial secondary messenger cyclic dinucleotides. It was first discovered in the Vibrio cholerae seventh pandemic strains and is involved in efficient intestinal colonization and chemotaxis regulation. Phosphodiesterases (PDEs) that degrade 3′3′-cGAMP play important regulatory roles in the relevant signaling pathways, and a previous study has identified three PDEs in V. cholerae, namely, V-cGAP1, V-cGAP2, and V-cGAP3, functioning in 3′3′-cGAMP degradation. We report the crystal structure, biochemical, and structural analyses of V-cGAP3, providing a foundation for understanding the mechanism of 3′3′-cGAMP degradation and regulation in general. Our crystal and molecular dynamic (MD)-simulated structures revealed that V-cGAP3 contains tandem HD-GYP domains within its N- and C-terminal domains, with similar three-dimensional topologies despite their low-sequence identity. Biochemical and structural analyses showed that the N-terminal domain plays a mechanism of positive regulation for the catalytic C-terminal domain. We also demonstrated that the other homologous Vibrio PDEs, V-cGAP1/2, likely function via a similar mechanism.