Publications (70)571.19 Total impact
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Article: Identification of c-di-GMP derivatives resistant to an EAL domain phosphodiesterase.
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ABSTRACT: The bacterial second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, is the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications to c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analog is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl-phosphate modified derivative of c-di-GMP containing 2'-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analog is also resistant to EAL domain catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling.Biochemistry 12/2012; · 3.42 Impact Factor -
Article: The bacterial second messenger c-di-GMP: probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs.
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ABSTRACT: The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs.Organic & Biomolecular Chemistry 10/2012; · 3.70 Impact Factor -
Article: IBI series winner. Student-directed discovery of the plant microbiome and its products.
Science 10/2012; 338(6106):485-6. · 31.20 Impact Factor -
Article: Structural and biochemical characterization of linear dinucleotide analogues bound to the c-di-GMP-I aptamer.
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ABSTRACT: The cyclic dinucleotide c-di-GMP regulates lifestyle transitions in many bacteria, such as the change from a free motile state to a biofilm-forming community. Riboswitches that bind this second messenger are important downstream targets in this bacterial signaling pathway. The breakdown of c-di-GMP in the cell is accomplished enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding riboswitches must be able to discriminate between their cognate cyclic ligand and linear dinucleotides in order to be selective biological switches. It has been reported that the c-di-GMP-I riboswitch binds c-di-GMP 5 orders of magnitude better than the linear pGpG, but the cause of this large energetic difference in binding is unknown. Here we report binding data and crystal structures of several linear c-di-GMP analogues in complex with the c-di-GMP-I riboswitch. These data reveal the parameters for phosphate recognition and the structural basis of linear dinucleotide binding to the riboswitch. Additionally, the pH dependence of binding shows that exclusion of pGpG is not due to the additional negative charge on the ligand. These data reveal principles that, along with published work, will contribute to the design of c-di-GMP analogues with properties desirable for use as chemical tools and potential therapeutics.Biochemistry 12/2011; 51(1):425-32. · 3.42 Impact Factor -
Article: Minimal transition state charge stabilization of the oxyanion during peptide bond formation by the ribosome.
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ABSTRACT: Peptide bond formation during ribosomal protein synthesis involves an aminolysis reaction between the aminoacyl α-amino group and the carbonyl ester of the growing peptide via a transition state with a developing negative charge, the oxyanion. Structural and molecular dynamic studies have suggested that the ribosome may stabilize the oxyanion in the transition state of peptide bond formation via a highly ordered water molecule. To biochemically investigate this mechanistic hypothesis, we estimated the energetic contribution to catalytic charge stabilization of the oxyanion using a series of transition state mimics that contain different charge distributions and hydrogen bond potential on the functional group mimicking the oxyanion. Inhibitors containing an oxyanion mimic that carried a neutral charge and a mimic that preserved the negative charge but could not form hydrogen bonds had less than a 3-fold effect on inhibitor binding affinity. These observations argue that the ribosome provides minimal transition state charge stabilization to the oxyanion during peptide bond formation via the water molecule. This is in contrast to the substantial level of oxyanion stabilization provided by serine proteases. This suggests that the oxyanion may be neutralized via a proton shuttle, resulting in an uncharged transition state.Biochemistry 12/2011; 50(48):10491-8. · 3.42 Impact Factor -
Article: Analysis of enzymatic transacylase Brønsted studies with application to the ribosome.
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ABSTRACT: Preferential binding of an enzyme to the transition state relative to the ground state is a key strategy for enzyme catalysis. When there is a difference between the ground and transition state charge distributions, enzymes maximize electrostatic interactions to achieve this enhanced transition state binding. Although the transition state is difficult to observe directly by structural methods, the chemical details of this transient species can be characterized by studies of substituent effects (Brønsted, Hammett, Swain-Scott, etc.) and isotope effects. Brønsted analysis can provide an estimate of transition state charges for the nucleophile and leaving group of a reaction. This Account will discuss the theoretical basis of Brønsted analysis and describe its practical application to the study of transacylase enzyme systems including the peptidyl transferase reaction of the ribosome. The Brønsted coefficient is derived from the linear free energy relationship (LFER) that correlates the acidity (pK(a)) of a reactive atom to the log of its rate constant. The Brønsted coefficient establishes the change in atomic charge as the reaction proceeds from the ground state to the transition state. Bonding events alter the electrostatics of atoms and the extent of bonding can be extrapolated from transition state charges. Therefore, well-defined nucleophile and leaving group transition state charges limit the number of mechanisms that are consistent with a particular transition state. Brønsted results are most informative when interpreted in the context of other mechanistic data, especially for enzymatic studies where an active site may promote a transition state that differs significantly from a prediction based on uncatalyzed solution reactions. Here we review Brønsted analyses performed on transacylases to illustrate how these data enhanced the enzymatic mechanistic studies. Through a systematic comparison of five enzymes, we reveal a wide spectrum of Brønsted values that are possible for what otherwise appear to be similar chemical reactions. The variations in the Brønsted coefficients predict different transition states for the various enzymes. This Account explores an overriding theme in the enzymatic mechanisms that catalysis enhances commensurate bond formation and proton abstraction events. The extent of the two bonding events in relationship to each other can be inferred from the Brønsted coefficient. When viewed in the context of recent ribosomal studies, this interpretation provides mechanistic insights into peptide bond formation.Accounts of Chemical Research 11/2011; 45(4):495-503. · 21.64 Impact Factor -
Article: The chemical versatility of RNA.
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ABSTRACT: The ability of RNA to both store genetic information and catalyse chemical reactions has led to the hypothesis that it predates DNA and proteins. While there is no doubt that RNA is capable of storing the genetic information of a primitive organism, only two classes of reactions-phosphoryl transfer and peptide bond formation-have been observed to be catalysed by RNA in nature. However, these naturally occurring ribozymes use a wide range of catalytic strategies that could be applied to other reactions. Furthermore, RNA can bind several cofactors that are used by protein enzymes to facilitate a wide variety of chemical processes. Despite its limited functional groups, these observations indicate RNA is a versatile molecule that could, in principle, catalyse the myriad reactions necessary to sustain life.Philosophical Transactions of The Royal Society B Biological Sciences 10/2011; 366(1580):2929-35. · 6.40 Impact Factor -
Article: Biodegradation of polyester polyurethane by endophytic fungi.
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ABSTRACT: Bioremediation is an important approach to waste reduction that relies on biological processes to break down a variety of pollutants. This is made possible by the vast metabolic diversity of the microbial world. To explore this diversity for the breakdown of plastic, we screened several dozen endophytic fungi for their ability to degrade the synthetic polymer polyester polyurethane (PUR). Several organisms demonstrated the ability to efficiently degrade PUR in both solid and liquid suspensions. Particularly robust activity was observed among several isolates in the genus Pestalotiopsis, although it was not a universal feature of this genus. Two Pestalotiopsis microspora isolates were uniquely able to grow on PUR as the sole carbon source under both aerobic and anaerobic conditions. Molecular characterization of this activity suggests that a serine hydrolase is responsible for degradation of PUR. The broad distribution of activity observed and the unprecedented case of anaerobic growth using PUR as the sole carbon source suggest that endophytes are a promising source of biodiversity from which to screen for metabolic properties useful for bioremediation.Applied and environmental microbiology 09/2011; 77(17):6076-84. · 3.69 Impact Factor -
Article: glmS Riboswitch binding to the glucosamine-6-phosphate α-anomer shifts the pKa toward neutrality.
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ABSTRACT: The glmS riboswitch regulates gene expression through a self-cleavage activity. The reaction is catalyzed with the assistance of the metabolite cofactor glucosamine-6-phosphate (GlcN6P), whose amino group is proposed to serve as the general acid during the reaction. This reaction is pH-dependent with a pK(a) that is lower than the observed pK(a) for the amine of GlcN6P in solution. GlcN6P, like other pyranose sugars, undergoes spontaneous and rapid interconversion between the α and β anomers at the C1 position. Here we demonstrate by NMR that the Bacillus anthracis glmS riboswitch selectively binds the α-anomer of GlcN6P with a maximum binding affinity of 0.36 mM and that binding is pH-dependent. We also report that the anomeric ratio between α and β is pH-dependent and the pK(a)s of the two amines differ by 0.5 pH units, α being the higher of the two (pK(a)=8.3). The pH dependence of binding reveals a pK(a) of 6.7, suggesting that the glmS RNA reduces the pK(a) of the GlcN6P amine by 1.6 units in the ground state. We reevaluated previously obtained kinetic data and found the reaction pK(a) is 6.9, within error of the binding data. The data support a model where the reaction pK(a) corresponds to that of the GlcN6P amine. This observation has broader relevance for considering how the microenvironment of an RNA, despite its anionic character, can reduce the pK(a)s of functional groups for use in catalysis.Biochemistry 08/2011; 50(33):7236-42. · 3.42 Impact Factor -
Article: Differential analogue binding by two classes of c-di-GMP riboswitches.
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ABSTRACT: The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism bacteria have evolved to sense environmental cues and translate these signals into phenotypic changes uses the second messenger signaling molecule, cyclic diguanosine monophosphate (c-di-GMP). In addition to several classes of protein receptors, two classes of c-di-GMP-binding riboswitches (class I and class II) have been identified as downstream targets of the second messenger in this signaling pathway. The crystal structures of both riboswitch classes bound to c-di-GMP were previously reported. Here, we further investigate the mechanisms that RNA has evolved for recognition and binding of this second messenger. Using a series of c-di-GMP analogues, we probed the interactions made in the RNA-ligand complex for both classes of riboswitches to identify the most critical elements of c-di-GMP for binding. We found that the structural features of c-di-GMP required for binding differ between these two effectors and that the class II riboswitch is much less discriminatory in ligand binding than the class I riboswitch. These data suggest an explanation for the predicted preferential use of the class I motif over the class II motif in the c-di-GMP signaling pathway.Journal of the American Chemical Society 08/2011; 133(39):15578-92. · 9.91 Impact Factor -
Article: A two-step chemical mechanism for ribosome-catalysed peptide bond formation.
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ABSTRACT: The chemical step of natural protein synthesis, peptide bond formation, is catalysed by the large subunit of the ribosome. Crystal structures have shown that the active site for peptide bond formation is composed entirely of RNA. Recent work has focused on how an RNA active site is able to catalyse this fundamental biological reaction at a suitable rate for protein synthesis. On the basis of the absence of important ribosomal functional groups, lack of a dependence on pH, and the dominant contribution of entropy to catalysis, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2'-hydroxyl of the peptidyl-transfer RNA and a Brønsted coefficient near zero have been taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation, based on analysis of the kinetic isotope effect at five positions within the reaction centre of a peptidyl-transfer RNA mimic. Our results indicate that in contrast to the uncatalysed reaction, formation of the tetrahedral intermediate and proton transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike in previous proposals, the reaction is not fully concerted; instead, breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.Nature 08/2011; 476(7359):236-9. · 36.28 Impact Factor -
Article: Structural basis of differential ligand recognition by two classes of bis-(3'-5')-cyclic dimeric guanosine monophosphate-binding riboswitches.
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ABSTRACT: The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway regulates biofilm formation, virulence, and other processes in many bacterial species and is critical for their survival. Two classes of c-di-GMP-binding riboswitches have been discovered that bind this second messenger with high affinity and regulate diverse downstream genes, underscoring the importance of RNA receptors in this pathway. We have solved the structure of a c-di-GMP-II riboswitch, which reveals that the ligand is bound as part of a triplex formed with a pseudoknot. The structure also shows that the guanine bases of c-di-GMP are recognized through noncanonical pairings and that the phosphodiester backbone is not contacted by the RNA. Recognition is quite different from that observed in the c-di-GMP-I riboswitch, demonstrating that at least two independent solutions for RNA second messenger binding have evolved. We exploited these differences to design a c-di-GMP analog that selectively binds the c-di-GMP-II aptamer over the c-di-GMP-I RNA. There are several bacterial species that contain both types of riboswitches, and this approach holds promise as an important tool for targeting one riboswitch, and thus one gene, over another in a selective fashion.Proceedings of the National Academy of Sciences 05/2011; 108(19):7757-62. · 9.68 Impact Factor -
Article: Interactions of the c-di-GMP riboswitch with its second messenger ligand.
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ABSTRACT: The c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] riboswitch is a macromolecular target in the c-di-GMP second messenger signalling pathway. It regulates many genes related to c-di-GMP metabolism as well as genes involved in bacterial motility, virulence and biofilm formation. The riboswitch makes asymmetric contacts to the bases and phosphate backbone of this symmetric dinucleotide. The phylogenetics suggested and mutagenesis has confirmed that this is a flexible motif where variants can make alternative interactions with each of the guanine bases of c-di-GMP. A mutant riboswitch has been designed that can bind a related molecule, c-di-AMP, confirming the most important contacts made to the ligand. The binding kinetics reveal that this is a kinetically controlled riboswitch and mutations to the riboswitch lead to increases in the off-rate. This riboswitch is therefore flexible in sequence as well as kinetic properties.Biochemical Society Transactions 04/2011; 39(2):647-51. · 3.71 Impact Factor -
Article: Structural basis of cooperative ligand binding by the glycine riboswitch.
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ABSTRACT: The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 Å crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.Chemistry & biology 03/2011; 18(3):293-8. · 6.52 Impact Factor -
Article: The 2'-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.
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ABSTRACT: The ribosome accelerates the rate of peptidyl transfer by >10(6)-fold relative to the background rate. A widely accepted model for this rate enhancement invokes entropic effects whereby the ribosome and the 2'-OH of the peptidyl-tRNA substrate precisely position the reactive moieties through an extensive network of hydrogen bonds that allows proton movement through them. Some studies, however, have called this model into question because they find the 2'-OH of the peptidyl-tRNA to be dispensable for catalysis. Here, we use an in vitro reconstituted translation system to resolve these discrepancies. We find that catalysis is at least 100-fold slower with the dA76-substituted peptidyl-tRNA substrate and that the peptidyl transferase centre undergoes a slow inactivation when the peptidyl-tRNA lacks the 2'-OH group. Additionally, the 2'-OH group was found to be critical for EFTu binding and peptide release. These findings reconcile the conflict in the literature, and support a model where interactions between active site residues and the 2'-OH of A76 of the peptidyl-tRNA are pivotal in orienting substrates in this active site for optimal catalysis.The EMBO Journal 01/2011; 30(12):2445-53. · 9.20 Impact Factor -
Article: Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch.
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ABSTRACT: The glycine riboswitch has a tandem dual aptamer configuration, where each aptamer is a separate ligand-binding domain, but the aptamers function together to bind glycine cooperatively. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and noncooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing, and native gel-shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Furthermore, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results provide insight into cooperative ligand binding in RNA macromolecules.RNA 01/2011; 17(1):74-84. · 5.09 Impact Factor -
Article: Structural and biochemical determinants of ligand binding by the c-di-GMP riboswitch .
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ABSTRACT: The bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that the most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high-resolution (2.3 A) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3'-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.Biochemistry 08/2010; 49(34):7351-9. · 3.42 Impact Factor -
Article: Transition states of uncatalyzed hydrolysis and aminolysis reactions of a ribosomal P-site substrate determined by kinetic isotope effects.
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ABSTRACT: The ester bond of peptidyl-tRNA undergoes nucleophilic attack in solution and when catalyzed by the ribosome. To characterize the uncatalyzed hydrolysis reaction, a model of peptide release, the transition state structure for hydrolysis of a peptidyl-tRNA mimic was determined. Kinetic isotope effects were measured at several atoms that potentially undergo a change in bonding in the transition state. Large kinetic isotope effects of carbonyl (18)O and alpha-deuterium substitutions on uncatalyzed hydrolysis indicate the transition state is nearly tetrahedral. Kinetic isotope effects were also measured for aminolysis by hydroxylamine to study a reaction similar to the formation of a peptide bond. In contrast to hydrolysis, the large leaving group (18)O isotope effect indicates the C-O3' bond has undergone significant scission in the transition state. The smaller carbonyl (18)O and alpha-deuterium effects are consistent with a later transition state. The assay developed here can also be used to measure isotope effects for the ribosome-catalyzed reactions. These uncatalyzed reactions serve as a basis for determining what aspects of the transition states are stabilized by the ribosome to achieve a rate enhancement.Biochemistry 04/2010; 49(18):3868-78. · 3.42 Impact Factor -
Article: Plasticity of the RNA kink turn structural motif.
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ABSTRACT: The kink turn (K-turn) is an RNA structural motif found in many biologically significant RNAs. While most examples of the K-turn have a similar fold, the crystal structure of the Azoarcus group I intron revealed a novel RNA conformation, a reverse kink turn bent in the direction opposite that of a consensus K-turn. The reverse K-turn is bent toward the major grooves rather than the minor grooves of the flanking helices, yet the sequence differs from the K-turn consensus by only a single nucleotide. Here we demonstrate that the reverse bend direction is not solely defined by internal sequence elements, but is instead affected by structural elements external to the K-turn. It bends toward the major groove under the direction of a tetraloop-tetraloop receptor. The ability of one sequence to form two distinct structures demonstrates the inherent plasticity of the K-turn sequence. Such plasticity suggests that the K-turn is not a primary element in RNA folding, but instead is shaped by other structural elements within the RNA or ribonucleoprotein assembly.RNA 02/2010; 16(4):762-8. · 5.09 Impact Factor -
Article: Structural basis of ligand binding by a c-di-GMP riboswitch.
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ABSTRACT: The second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 A resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.Nature Structural & Molecular Biology 11/2009; 16(12):1218-23. · 12.71 Impact Factor
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- Biochemistry (7)
- Biochemistry (7)
- RNA (5)
- Nucleic Acids Research (4)
- RNA (4)
Institutions
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2002–2012
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Yale University
- • Department of Chemistry
- • Department of Molecular Biophysics and Biochemistry
New Haven, CT, USA
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