The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans' capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J-coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac3, calculated from MD simulations. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by ß-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a ß-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans capsular polysaccharide and shed exopolysaccharide functions both as a key virulence factor and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this combined NMR and MD study, experimentally determined NOEs and J-couplings support a structure of the synthetic decasaccharide, GXM10-Ac3, obtained by MD. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is characterized by a 6-residue α-mannan backbone repeating unit, consisting of a triad of α-(1→3)-mannoses, modified by ß-(1→2)-xyloses on the first two mannoses and a ß-(1→2)-glucuronic acid on the third mannose. The combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with xylose/glucuronic acid branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This experimentally consistent GXM model displays high flexibility while maintaining a structural identity, yielding new insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

Glycan Stability and Flexibility: Thermodynamic and Kinetic Characterization of Nonconventional Hydrogen Bonding in Lewis Antigens.

We provide evidence for CH-based nonconventional hydrogen bonds (H-bonds) for 10 Lewis antigens and two of their rhamnose analogues. We also characterize the thermodynamics and kinetics of the H-bonds in these molecules and present a plausible explanation for the presence of nonconventional H-bonds in Lewis antigens. Using an alternative method to simultaneously fit a series of temperature-dependent fast exchange nuclear magnetic resonance (NMR) spectra, we determined that the H-bonded conformation is favored by ∼1 kcal/mol over the non-H-bonded conformation. Additionally, a comparison of temperature-dependent 13C linewidths in various Lewis antigens and the two rhamnose analogues reveals H-bonds between the carbonyl oxygen of the N-acetyl group of N-acetylglucosamine and the OH2 group of galactose/fucose. The data presented herein provide insight into the contribution of nonconventional H-bonding to molecular structure and could therefore be used for the rational design of therapeutics.

The Incorporation of Labile Protons into Multidimensional NMR Analyses: Glycan Structures Revisited.

Glycan structures are often stabilized by a repertoire of hydrogen-bonded donor/acceptor groups, revealing longer-lived structures that could represent biologically relevant conformations. NMR provides unique data on these hydrogen-bonded networks from multidimensional experiments detecting cross-peaks resulting from through-bond (TOCSY) or through-space (NOESY) interactions. However, fast OH/H2O exchange, and the spectral proximity among these NMR resonances, hamper the use of glycans' labile protons in such analyses; consequently, studies are often restricted to aprotic solvents or supercooled aqueous solutions. These nonphysiological conditions may lead to unrepresentative structures or to probing a small subset of accessible conformations that may miss "active" glycan conformations. Looped, projected spectroscopy (L-PROSY) has been recently shown to substantially enhance protein NOESY and TOCSY cross-peaks, for 1Hs that undergo fast exchange with water. This study shows that even larger enhancements can be obtained for rapidly exchanging OHs in saccharides, leading to the retrieval of previously undetectable 2D TOCSY/NOESY cross-peaks with nonlabile protons. After demonstrating ≥300% signal enhancements on model monosaccharides, these experiments were applied at 1 GHz to elucidate the structural network adopted by a sialic acid homotetramer, used as a model for α,2-8 linked polysaccharides. High-field L-PROSY NMR enabled these studies at higher temperatures and provided insight previously unavailable from lower-field NMR investigations on supercooled samples, involving mostly nonlabile nuclei. Using L-PROSY's NOEs and other restraints, a revised structural model for the homotetramer was obtained combining rigid motifs and flexible segments, that is well represented by conformations derived from 40 µs molecular dynamics simulations.

Characterization of the ß-KDO Transferase KpsS, the Initiating Enzyme in the Biosynthesis of the Lipid Acceptor for Escherichia coli Polysialic Acid.

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the ß-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a ß-KDO linkage.

Chemical structure and genetic organization of the E. coli O6:K15 capsular polysaccharide.

Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gram-negative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identified as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1 → 5)-α-KDOp-(2 → partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.

The ß-reducing end in α(2-8)-polysialic acid constitutes a unique structural motif.

Over the years, structural characterizations of α(2-8)-polysialic acid (polySia) in solution have produced inconclusive results. Efforts for obtaining detailed information in this important antigen have focused primarily on the α-linked residues and not on the distinctive characteristics of the terminal ones. The thermodynamically preferred anomeric configuration for the reducing end of sialic acids is ß, which has the [I]CO2- group equatorial and the OH ([I]OH2) axial, while for all other residues the CO2- group is axial. We show that this purportedly minor difference has distinct consequences for the structure of α(2-8)-polySia near the reducing end, as the ß configuration places the [I]OH2 in a favorable position for the formation of a hydrogen bond with the carboxylate group of the following residue ([II]CO2-). Molecular dynamics (MD) simulations predicted the hydrogen bond, which we subsequently directly detected by NMR. The combination of MD and residual dipolar couplings shows that the net result for the structure of Sia2-ßOH is a stable conformation with well-defined hydration and charge patterns, and consistent with experimental NOE-based hydroxyl and aliphatic inter-proton distances. Moreover, we provide evidence that this distinct conformation is preserved on Sia oligosaccharides, thus constituting a motif that determines the structure and dynamics of α(2-8)-polySia for at least the first two residues of the polymer. We suggest the hypothesis that this structural motif sheds light on a longtime puzzling observation for the requirement of 10 residues of α(2-8)-polySia in order to bind effectively to specific antibodies, about four units more than for analogous cases.

Glycan OH Exchange Rate Determination in Aqueous Solution: Seeking Evidence for Transient Hydrogen Bonds.

Hydrogen bonds (Hbonds) are important stabilizing forces in biomolecules. However, for glycans in aqueous solution, direct NMR detection of Hbonds is elusive because of their transient nature. Here, we present Isotope-based Natural-abundance TOtal correlation eXchange SpectroscopY (INTOXSY), a new 1H-13C heteronuclear single quantum coherence-total correlation spectroscopy based method, to extract OH groups' exchange rate constants (kex) for molecules in natural 13C abundance and show that OH Hbonds can be inferred from "slower" H/D kex. We evaluate kex measured with INTOXSY in light of those extracted with line-shape analysis. Subsequently, we use a set of common glycans to establish a kex reference basis set and to infer the existence of transient Hbonds involving OH donor groups. Then, we report kex values for a series of mono- and disaccharides, as well as for oligosaccharides sialyl Lewis X and ß-cyclodextrin, and compare the results with those from the reference set to extract Hbond information. Finally, we utilize NMR experimental data in conjunction with molecular dynamics simulations to establish donor and acceptor Hbond pairs. Our exchange rate measurements indicate that OH/OD exchange rates, kHD, values <10 s-1 are consistent with transient Hbond OH groups and potential acceptor groups can be uncovered through MD simulations.

Uncovering Nonconventional and Conventional Hydrogen Bonds in Oligosaccharides through NMR Experiments and Molecular Modeling: Application to Sialyl Lewis-X.

We describe the direct NMR detection of a C-H···O nonconventional hydrogen bond (Hbond) and provide experimental and theoretical evidence for conventional Hbonds in the pentasaccharide sialyl Lewis-X (sLe(X)-5) between 5 and 37 °C in water. Extensive NMR structural studies together with molecular dynamics simulations offer strong evidence for significant local dynamics in the Le(X) core and for previously undetected conventional Hbonds in rapid equilibrium that modulate structure. These NMR studies also showed temperature-dependent (1)H and (13)C line broadening. The resulting model emerging from this study is more complex than a simple rigid core description of Le(X)-like molecules and improves our understanding of stabilizing interactions in glycans.

NMR of glycans: shedding new light on old problems.

The diversity in molecular arrangements and dynamics displayed by glycans renders traditional NMR strategies, employed for proteins and nucleic acids, insufficient. Because of the unique properties of glycans, structural studies often require the adoption of a different repertoire of tailor-made experiments and protocols. We present an account of recent developments in NMR techniques that will deepen our understanding of structure-function relations in glycans. We open with a survey and comparison of methods utilized to determine the structure of proteins, nucleic acids and carbohydrates. Next, we discuss the structural information obtained from traditional NMR techniques like chemical shifts, NOEs/ROEs, and coupling-constants, along with the limitations imposed by the unique intrinsic characteristics of glycan structure on these approaches: flexibility, range of conformers, signal overlap, and non-first-order scalar (strong) coupling. Novel experiments taking advantage of isotopic labeling are presented as an option for overcoming spectral overlap and raising sensitivity. Computational tools used to explore conformational averaging in conjunction with NMR parameters are described. In addition, recent developments in hydroxyl detection and hydrogen bond detection in protonated solvents, in contrast to traditional sample preparations in D2O for carbohydrates, further increase the tools available for both structure information and chemical shift assignments. We also include previously unpublished data in this context. Accurate determination of couplings in carbohydrates has been historically challenging due to the common presence of strong-couplings. We present new strategies proposed for dealing with their influence on NMR signals. We close with a discussion of residual dipolar couplings (RDCs) and the advantages of using (13)C isotope labeling that allows gathering one-bond (13)C-(13)C couplings with a recently improved constant-time COSY technique, in addition to the commonly measured (1)H-(13)C RDCs.

Accurate determinations of one-bond 13C-13C couplings in 13C-labeled carbohydrates.

Carbon plays a central role in the molecular architecture of carbohydrates, yet the availability of accurate methods for (1)D(CC) determination has not been sufficiently explored, despite the importance that such data could play in structural studies of oligo- and polysaccharides. Existing methods require fitting intensity ratios of cross- to diagonal-peaks as a function of the constant-time (CT) in CT-COSY experiments, while other methods utilize measurement of peak separation. The former strategies suffer from complications due to peak overlap, primarily in regions close to the diagonal, while the latter strategies are negatively impacted by the common occurrence of strong coupling in sugars, which requires a reliable assessment of their influence in the context of RDC determination. We detail a (13)C-(13)C CT-COSY method that combines a variation in the CT processed with diagonal filtering to yield (1)J(CC) and RDCs. The strategy, which relies solely on cross-peak intensity modulation, is inspired in the cross-peak nulling method used for J(HH) determinations, but adapted and extended to applications where, like in sugars, large one-bond (13)C-(13)C couplings coexist with relatively small long-range couplings. Because diagonal peaks are not utilized, overlap problems are greatly alleviated. Thus, one-bond couplings can be determined from different cross-peaks as either active or passive coupling. This results in increased accuracy when more than one determination is available, and in more opportunities to measure a specific coupling in the presence of severe overlap. In addition, we evaluate the influence of strong couplings on the determination of RDCs by computer simulations. We show that individual scalar couplings are notably affected by the presence of strong couplings but, at least for the simple cases studied, the obtained RDC values for use in structural calculations were not, because the errors introduced by strong couplings for the isotropic and oriented phases are very similar and therefore cancel when calculating the difference to determine (1)D(CC) values.

Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry.

Oomycetes such as Phytophthora sojae employ effector proteins that enter plant cells to facilitate infection. Entry of some effector proteins is mediated by RxLR motifs in the effectors and phosphoinositides (PIP) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effectors involved in PIP recognition. We have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that Avh5 is helical in nature, with a long N-terminal disordered region. NMR titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate, directly identified the ligand-binding residues. A C-terminal lysine-rich helical region (helix 2) was the principal lipid-binding site, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Mutations in the RFLR motif or in the basic region both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. Based on these findings, we propose a model where Avh5 interacts with PtdIns(3)P through its C terminus, and by binding of the RFLR motif, which promotes host cell entry.

The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides.

TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.

Backbone (1)H, (15)N, and (13)C resonance assignments and secondary structure of the Tollip CUE domain.

The Toll-interacting protein (Tollip) is a negative regulator of the Toll-like receptor (TLR)-mediated inflammation response. Tollip is a modular protein that contains an Nterminal Tom1-binding domain (TBD), a central conserved domain 2 (C2), and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Here, we report the sequence-specific backbone (1)H, (15)N, and (13)C assignments of the human Tollip CUE domain. The CUE domain was found to be a stable dimer as determined by size-exclusion chromatography and molecular crosslinking studies. Analysis of the backbone chemical shift data indicated that the CUE domain exhibits three helical elements corresponding to 52% of the protein backbone. Circular dichroism spectrum analysis confirmed the helical nature of this domain. Comparison of the location of these helical regions with those reported for yeast CUE domains suggest differences in length for all helical elements. We expect the structural analysis presented here will be the foundation for future studies on the biological significance of the Tollip CUE domain, its molecular interactions, and the mechanisms that modulate its function during the inflammatory response.

Structural and membrane binding properties of the prickle PET domain.

The planar cell polarity (PCP) pathway is required for fetal tissue morphogenesis as well as for maintenance of adult tissues in animals as diverse as fruit flies and mice. One of the key members of this pathway is Prickle (Pk), a protein that regulates cell movement through its association with the Dishevelled (Dsh) protein. Pk presents three LIM domains and a PET domain of unknown structure and function. Both the PET and LIM domains control membrane targeting of Dsh, which is necessary for Dsh function in the PCP pathway. Here, we show that the PET domain is monomeric and presents a nonglobular conformation with some properties of intrinsically disordered proteins. The PET domain adopts a helical conformation in the presence of 2,2,2-trifluoroethanol (TFE), a solvent known to stabilize hydrogen bonds within the polypeptide backbone, as analyzed by circular dichroism (CD) and NMR spectroscopy. Furthermore, we found that the conserved and single tryptophan residue in PET, Trp 536, moves to a more hydrophobic environment when accompanied with membrane penetration and that the protein becomes more helical in the presence of lipid micelles. The presence of LIM domains, downstream of PET, increases protein folding, thermostability, and tolerance to limited proteolysis. In addition, pull-down and tryptophan fluorescence analyses suggest that the LIM domains physically interact to regulate membrane penetration of the PET domain. The findings reported here favor a model where the PET domain is engaged in Pk membrane insertion, whereas the LIM domains modulate this function.

Extracellular structure of polysialic acid explored by on cell solution NMR.

The capsular polysaccharide of the pathogens Neisseria meningitidis serogroup B and of Escherichia coli K1, alpha(2 --> 8) polysialic acid (PSA), is unusual, because when injected into adult humans, it generates little or no antibody. In contrast, people infected with these pathogens generate specific serum antibodies. A structural study on cells is used to address this anomaly by characterizing antigen structures in vivo. We introduce on cell multidimensional solution NMR spectroscopy for direct observation of PSA on E. coli bacteria. Using 13C,15N-labeled PSA, we applied a combination of heteronuclear NMR methods, such as heteronuclear single quantum coherence, HNCA, and HNCO, in vivo. Analysis reveals that free and cell-bound PSA are structurally similar, indicating that the poor immunogenicity of PSA is not due to major structural differences between cells and purified PSA. The 13C linewidths of PSA on cells are 2 to 3 times larger than the corresponding ones in free PSA. The possible implications of the differences between free and on cell PSA are discussed. In addition, we demonstrate the suitability of the method for in vivo kinetic studies.

X-ray, NMR, and mutational studies of the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction.

GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg2+ (Gln-123), the catalytic base (His-124), and three anionic residues. This loop is not ordered in the X-ray structure of the free enzyme due to dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selective exchange broadening of the imidazole nitrogen resonances of His-124 (k(ex) = 6.6 x 10(4) s(-1)). The structure of the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6 in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loop L6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole N epsilon of His-124 to be unprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metal ions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of a metal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 10(3.4)-fold and largely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold and restores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shifts the conformational equilibrium to the open form moving loop L6 out of the active site, thus permitting direct access of the specific base hydroxide from the solvent. In the proposed dissociative transition state, which occurs in the closed, active conformation of the enzyme, the partial negative charge of the GDP leaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to the beta-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transition state is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizing the transition state.

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction.

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.

Mutational, structural, and kinetic evidence for a dissociative mechanism in the GDP-mannose mannosyl hydrolase reaction.

GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-alpha-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP.Tris(+) complex [Gabelli, S. B., et al. (2004) Structure 12, 927-935], the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg(2+). As determined by the effects of site-specific mutants on k(cat), these components contribute factors of 24-, 100-, 309-, 24-, and >/=10(5)-fold, respectively, to catalysis. Both R37 and Y103 bind the beta-phosphate of GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants on k(cat), indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects on K(m). Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an alpha-d-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased k(cat) by factors of 10(2.1) and 10(2.6), respectively, for the hydrolysis of GDP-alpha-d-mannose, and showed smaller effects on K(m), suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-alpha-d-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease in k(cat) and a smaller, 2.5-fold increase in K(m). The D22A and D22N mutations further decreased the k(cat) with GDP-2F-alpha-d-mannose to values similar to those found with GDP-alpha-d-mannose, and decreased the K(m) of the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pK(a) of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10(2.2)-fold decrease in k(cat) and a 4.0-fold increase in K(m) at pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects on k(cat) and K(m) with either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex.

Half-of-the-sites binding of reactive intermediates and their analogues to 4-oxalocrotonate tautomerase and induced structural asymmetry of the enzyme.

4-Oxalocrotonate tautomerase (4-OT), a homohexameric enzyme, converts the unconjugated enone, 2-oxo-4-hexenedioate (1), to the conjugated enone, 2-oxo-3-hexenedioate (3), via a dienolic intermediate, 2-hydroxymuconate (2). Pro-1 serves as the general base, and both Arg-11 and Arg-39 function in substrate binding and catalysis in an otherwise hydrophobic active site. Although 4-OT exhibits hyperbolic kinetics and no structural asymmetry either by X-ray or by NMR, inactivation by two affinity labels showed half-site stoichiometry [Stivers, J. T., et al. (1996) Biochemistry 35, 803-813; Johnson, W. H., Jr., et al. (1997) Biochemistry 36, 15724-15732], and titration of the R39Q mutant with cis,cis-muconate showed negative cooperativity [Harris, T. K., et al. (1999) Biochemistry 38, 12343-12357]. To test for anticooperativity during catalysis, 4-OT was titrated with equilibrium mixtures (> or = 81% product) of the reactive dicarboxylate or monocarboxylate intermediates, 2 or 2-hydroxy-2,4-pentadienoate (4), respectively, in three types of NMR experiments: two-dimensional 1H-15N HSQC titrations of backbone NH and of Arg N epsilonH resonances and one-dimensional 15N NMR titrations of Arg N epsilon resonances. All titrations showed substoichiometric binding of the equilibrium mixtures to 3 +/- 1 sites per hexamer with apparent dissociation constants comparable to the Km values of the intermediates. Compound 4 also bound 1 order of magnitude less tightly at another site, suggesting negative cooperativity. Consistent with negative cooperativity, asymmetry of the resulting complexes at saturating levels of 2 and 4 is indicated by splitting of the backbone NH resonances of 11 residues and 10 residues of 4-OT, respectively. The dicarboxylate competitive inhibitor, (2E)-fluoromuconate (5), with a KI of 45 +/- 7 microM, also exhibited substoichiometric binding to 3 +/- 1 sites per hexamer, with a KD of 25 +/- 18 microM, and splitting of the backbone NH resonance of L8. The monocarboxylate inhibitors (2E)- (6) and (2Z)-2-fluoro-2,4-pentadienoate (7) showed much weaker binding (KD = 3.1 +/- 1.3 mM), as well as splitting of two and five backbone NH resonances, respectively, indicating asymmetry of the complexes. The N epsilon resonances of both Arg-11 and Arg-39 were shifted downfield, and that of Pro-1N was broadened by all ligands, consistent with the major catalytic roles of these residues. Structural pathways for the site-site interactions which result in negative cooperativity are proposed on the basis of the X-ray structures of free and affinity-labeled 4-OT. Selective resonance broadenings induced by the binding of inactive analogues and active intermediates indicate residues which may be mobilized during reversible ligand binding and during catalysis, respectively.