Various binuclear metal ion clusters and complexes have been reconstituted in crystalline human arginase I by removing the Mn2+2-cluster of the wild-type enzyme with metal chelators and subsequently soaking the crystalline apoenzyme in buffer solutions containing NiCl2 or ZnCl2. X-ray crystal structures of these metal ion variants are correlated with catalytic activity measurements that reveal differences resulting from metal ion substitution. Additionally, treatment of crystalline Mn2+2-human arginase I with Zn2+ reveals for the first time the structural basis for inhibition by Zn2+, which forms a carboxylate-histidine-Zn2+ triad with H141 and E277. The imidazole side chain of H141 is known to be hyper-reactive and its chemical modification or mutagenesis is known to similarly compromise catalysis. The reactive substrate analogue 2(S)-amino-6-boronohexanoic acid (ABH) binds as a tetrahedral boronate anion to Mn2+2, Co2+2, Ni2+2, and Zn2+2 clusters in human arginase I, and it can be stabilized by a third inhibitory Zn2+ ion coordinated by H141. Since ABH binds as an analogue of the tetrahedral intermediate and its flanking transition states in catalysis, this implies that the various metallosubstituted enzymes are capable of some level of catalysis with an actual substrate. Accordingly, we establish the following trend for turnover number (kcat) and catalytic efficiency (kcat/KM): Mn2+ > Ni2+ ≈ Co2+ ≫ Zn2+. Therefore, Mn2+ is required for optimal catalysis by human arginase I.
Earlier studies on electron transfer (ET) from the nitrogenase Fe protein to the MoFe protein concluded that the mechanism for ET changed during cooling from 25°C to 5°C, based on the observation that the rate constant for Fe protein to MoFe protein ET decreases strongly, with a non-linear Arrhenius plot. They further indicated that the ET was reversible, with complete ET at ambient but with an equilibrium constant near unity at 5°C. These studies were carried out with buffers having a strong temperature coefficient. We have examined the temperature variation in the kinetics of oxidation of the Fe protein by the MoFe protein at constant pH = 7.4 fixed by the buffer MOPS, which has a very small temperature coefficient. Using MOPS, we also observe temperature dependent ET rate constants, with non-linear Arrhenius plots. But, we find that ET is gated across the temperature range by a conformational change that involves the binding of numerous water molecules, consistent with an unchanging ET mechanism. Furthermore, there is no sKIE throughout the temperature range studied, again consistent with an unchanging mechanism In addition, the non-linear Arrhenius plots are explained by the change in heat capacity caused by the binding of waters in an invariant gating ET mechanism. Together, these observations contradict the idea of a change in ET mechanism with cooling. Finally, the extent of ET at constant pH does not change significantly with temperature, in contrast to the previously proposed change in ET equilibrium.
Rhodospirillum rubrumproduces 5-methylthioadenosine (MTA) from S-adenosylmethionine (SAM) in polyamine biosynthesis; however, R. rubrum lacks the classical methionine salvage pathway. Instead, MTA is converted to 5-methylthio-D-ribose 1-phosphate (MTR 1-P) and adenine; MTR 1-P is isomerized to 1-methylthio-D-xylulose 5-phosphate (MTXu 5-P) and reductively dethiomethylated to 1-deoxy-D-xylulose 5-phosphate (DXP), an intermediate in the nonmevalonate isoprenoid pathway (Erb et al. Nature Chem. Biol., in press). Dethiomethylation, a novel route to DXP, is catalyzed by MTXu 5-P methylsulfurylase. An active site Cys displaces the enolate of DXP from MTXu 5-P, generating a methyl disulfide intermediate.
Decorin binding protein A (DBPA) is an important lipoprotein from the bacterium Borrelia burgdorferi, the causative agent of Lyme disease. Absence of DBPA drastically reduces the pathogenic potential of the bacterium and biochemical evidence indicates DBPA’s interactions with the glycosaminoglycan (GAG) portion of decorin are crucial to its function. We have solved the solution structure of DBPA and studied DBPA’s interactions with various forms of GAGs. DBPA is determined to be a helical bundle protein consisting of five helices held together by a strong hydrophobic core. The structure also possesses a basic patch formed by portions of two helices and two flexible linkers. Low molecular weight heparin-induced chemical shift perturbations for residues in the region as well as increases in signal intensities of select residues in their presence confirm residues in the pocket are perturbed by heparin binding. Dermatan sulfate (DS) fragments, the dominant GAG type found on decorin, were shown to have lower affinity than heparin but are still capable of binding DBPA.
glycosaminoglycan; bacterial adhesin; GAG-protein interactions; solution NMR
Myotonic dystrophy type 1 (DM1) is a microsatellite expansion disorder caused by the aberrant expansion of CTG repeats in the 3’ untranslated region of the DMPK gene. When transcribed, the toxic RNA CUG repeats sequester RNA binding proteins, which leads to disease symptoms. The expanded CUG repeats can adopt a double-stranded structure, and targeting this helix is a therapeutic strategy for DM1. In order to better understand the 5’CUG/3’GUC motif, and how it may interact with proteins and small molecules, we designed a short CUG helix attached to a GAAA tetraloop/receptor in order to facilitate crystal packing. Here we report the highest resolution structure (1.95 Å) to date of a GAAA tetraloop/receptor and the CUG helix it was used to crystallize. Within the CUG helix, we identify two different forms of non-canonical U-U pairs and reconfirm that CUG repeats are essentially A-form. An analysis of all non-canonical U-U pairs in the context of CUG repeats revealed six different classes of conformations that the non-canonical U-U pairs are able to adopt.
Leishmania major peroxidase (LmP) exhibits both ascorbate and cytochrome c peroxidase activities. Our previous results illustrated that LmP has much higher activity against horse heart cytochrome c than ascorbate suggesting that cytochrome c may be the biologically important substrate. In order to elucidate the biological function of LmP, we have recombinantly expressed, purified and determined the 2.08Å crystal structure of Leishmania major cytochrome c (LmCytc). Like other cytochromes c LmCytc has an electropositive surface surrounding the exposed heme edge that serves as the docking site with redox partners. Kinetic assays performed with LmCytc and LmP show that LmCytc is a much better substrate for LmP than horse heart cytochrome c. Furthermore, unlike the well-studied yeast system, the reaction follows classic Michaelis-Menten kinetics and is sensitive to increasing ionic strength. Using the yeast co-crystal as a control, protein-protein docking was performed using Rosetta to develop a model for the binding of LmP and LmCytc. These results suggest that the biological function of LmP is to act as a cytochrome c peroxidase.
Peptide-triazole (PT) entry inhibitors prevent HIV-1 infection by blocking viral gp120 binding to both HIV-1 receptor and coreceptor on target cells. Here, we used all-atom explicit solvent molecular dynamics (MD) to propose a model for the encounter complex of the peptide-triazoles with gp120. Saturation Transfer Difference NMR (STD NMR) and single-site mutagenesis experiments were performed to test the simulation results. We found that docking of the peptide to a conserved patch of residues lining the “F43 pocket” of gp120 in a bridging sheet naïve gp120 conformation of the glycoprotein, led to a stable complex. This pose prevents formation of the bridging sheet minidomain, which is required for receptor/coreceptor binding, providing a mechanistic basis for dual-site antagonism of this class of inhibitors. Burial of the peptide triazole at gp120 inner/outer domain interface significantly contributed to complex stability and rationalizes the significant contribution of hydrophobic triazole groups to peptide potency. Both the simulation model and STD NMR experiments suggest that the I-X-W (where X=(2S, 4S)-4-(4-phenyl-1H-1, 2, 3-triazol-1-yl) pyrrolidine) tripartite hydrophobic motif in the peptide is the major contributor of contacts at the gp120/PT interface. Since the model predicts that the peptide Trp side chain hydrogen bonding with gp120 S375 contributes to stability of the PT/gp120 complex, we tested this prediction through analysis of peptide binding to gp120 mutant S375A. The results showed that a peptide triazole KR21 inhibits S375A with 20-fold less potency versus WT, consistent with predictions of the model. Overall, the PT/gp120 model provides a starting point for both rational design of higher affinity peptide triazoles and development of structure-minimized entry inhibitors that can trap gp120 into an inactive conformation and prevent infection.
Cytochrome c oxidase (CcO) reduces O2 to water via a series of proton coupled electron transfers generating a transmembrane electrochemical gradient. Coupling electron and proton transfer requires changing buried residues pKas at each stage in the reaction cycle. Heme a is a key cofactor in the CcO electron transfer chain. Mutation of Ser44 to Asp has been reported (Mills et al Biochemistry (2008) 47, 11499-11509), changing the hydrogen bond acceptor from His102, the Heme a axial ligand in Rhodobactor sphaeroides CcO. This adds an acidic residue to the CcO interior. The electrochemical behavior of Heme a in wild type and S44D CcO is compared using the continuum electrostatics program MCCE. The introduced, deeply buried Asp remains ionized at physiological pH only when the nearby heme is oxidized. Heme a reduction is now calculated to be strongly coupled to Asp proton binding, while with Ser44 it is weakly coupled to small protonation shifts at multiple sites, increasing the pH dependence in the mutant. At pH 7, the partially ionized Asp44 is calculated to lower the Heme redox potential by 50 mV as expected given the thermodynamics of coupled electron and proton transfers. This highlights an inconsistency in the experimental results where a low Asp pKa is found together with a stabilized reduced Heme. The stabilization of a model complex heme oxidation by a hydrogen bond to the axial His ligand calculated with Continuum Electrostatics and with Density Functional Theory was in good agreement.
cytochrome c oxidase; Heme a; MCCE; continuum electrostatics; Em; pKa; proton coupled electron transfer
Amyloid formation, a complex process involving many intermediate states, is proposed to be the driving force for amyloid-related toxicity in common degenerative diseases. Unfortunately, the details of this process have been obscured by the limitations in the methods that can follow this reaction in real-time. We show that alternative pathways of aggregation can be distinguished by using 19F NMR to monitor monomer consumption along with complementary measurements of fibrillogenesis. The utility of this technique is demonstrated by tracking amyloid formation in the diabetes-related islet amyloid polypeptide (IAPP). Using this technique, we show IAPP fibrillizes without an appreciable build up of non-fibrillar intermediates, in contrast to the well-studied Aβ and α-synuclein proteins. To further develop the usage of 19F NMR, we have tracked the influence of the polyphenolic amyloid inhibitor epigallocatechin gallate (EGCG) on the aggregation pathway. Polyphenols have been shown to strongly inhibit amyloid formation in many systems. However, spectroscopic measurements of amyloid inhibition by these compounds can be severely compromised by background signals and competitive binding with extrinsic probes. Using 19F NMR, we show that thioflavin T strongly competes with EGCG for binding sites on IAPP fibers. By comparing the rates of monomer consumption and fiber formation, we are able to show that EGCG stabilizes non-fibrillar large aggregates during fibrillogenesis.
IAPP; fluorine NMR; kinetics; mechanism; EGCG; inhibitor
The ability of Azotobacter vinelandii
NifIscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe-S cluster biogenesis. NifIscA is shown to bind one Fe(III) or one Fe(II) per homodimer and the spectroscopic and redox properties of both the Fe(III)- and Fe(II)-bound forms have been characterized using the UV-visible absorption, CD and VTMCD, EPR, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic intermediate-spin (S = 3/2) Fe(III) center (E/D = 0.33, D = 3.5 ± 1.5cm−1) that is most likely 5-coordinate with two or three cysteinate ligands and a rhombic high spin (S = 2) Fe(II) center (E/D = 0.28, D = 7.6 cm−1) with properties similar to reduced rubredoxins or rubredoxin variants with three cysteinate and one or two oxygenic ligands. Iron-bound NifIscA undergoes reversible redox cycling between the Fe(III)/Fe(II) forms with a midpoint potential of +36 ±15 mV at pH 7.8 (versus NHE). L-cysteine is effective in mediating release of free Fe(II) from both the Fe(II)- and Fe(III)-bound forms of NifIscA. Fe(III)-bound NifIscA was also shown to a competent iron source for in vitro NifS-mediated [2Fe-2S] cluster assembly on the N-terminal domain of NifU, but the reaction occurs via cysteine-mediated release of free Fe(II) rather than direct iron transfer. The proposed roles of A-type proteins in storing Fe under aerobic growth conditions and serving as iron donors for cluster assembly on U-type scaffold proteins or maturation of biological [4Fe-4S] centers are discussed in light of these results.
Hematopoietic tyrosine phosphatase (HePTP) regulates orthogonal MAP kinase signaling cascades by dephosphorylating both ERK and p38. HePTP recognizes a docking site (D-recruitment site, DRS) on its targets using a conserved N-terminal sequence motif (D-motif). Using solution NMR spectroscopy and isothermal titration calorimetry (ITC), we compare, for the first time, the docking interactions of HePTP with ERK2 and p38α. Our results demonstrate that ERK2/HePTP interactions primarily involve the D-motif, while a contiguous region called the kinase specificity motif (KIS) also plays a key role in p38α/HePTP interactions. D-motif/DRS interactions for the two kinases, while similar overall, do show some specific differences.
MAP kinase; ERK; p38; tyrosine phosphatase; HePTP; D-recruitment site; TROSY
The bacterial cell wall is essential to cell survival and is a major target of antibiotics. The main component of the bacterial cell wall is peptidoglycan, a cage-like macromolecule that preserves cellular integrity and maintains cell shape. The insolubility and heterogeneity of peptidoglycan pose a challenge to conventional structural analyses. Here we use solid-state NMR combined with specific isotopic labeling to probe a key structural feature of the Staphylococcus aureus peptidoglycan quantitatively and nondestructively. We observed that both the cell-wall morphology and the peptidoglycan structure are functions of growth stage in S. aureus synthetic medium (SASM). Specifically, S. aureus cells at stationary phase have thicker cell walls with non-uniformly thickened septa compared to cells in exponential phase and, remarkably, 12% (±2%) of the stems in their peptidoglycan do not have pentaglycine bridges attached. Mechanistically, we determined that these observations are triggered by the depletion of glycine in the nutrient medium, which is coincident with the start of the stationary phase, and that the production of the structurally altered peptidoglycan can be prevented by the addition of excess glycine. We also demonstrated that the structural changes primarily arise within newly synthesized peptidoglycan rather than through the modification of previously synthesized peptidoglycan. Collectively, our observations emphasize the plasticity in bacterial cell-wall assembly and the possibility to manipulate peptidoglycan structure with external stimuli.
bacterial cell wall; peptidoglycan; solid-state NMR; CPMAS; REDOR; nutrient status; growth stage
The mechanism of [4Fe-4S] cluster assembly on A-type Fe-S cluster assembly proteins, in general, and the specific role of NifIscA in the maturation of nitrogen fixation proteins are currently unknown. To address these questions, in vitro spectroscopic studies (UV–visible absorption/CD, resonance Raman and Mössbauer) have been used to investigate the mechanism of [4Fe-4S] cluster assembly on Azotobacter vinelandii
NifIscA, and the ability of NifIscA to accept clusters from NifU and to donate clusters to the apo form of the nitrogenase Fe-protein. The results show that NifIscA can rapidly and reversibly cycle between forms containing one [2Fe-2S]2+ and one [4Fe-4S]2+ cluster per homodimer via DTT-induced two-electron reductive coupling of two [2Fe-2S]2+ clusters and O2-induced [4Fe-4S]2+ oxidative cleavage. This unique type of cluster interconversion in response to cellular redox status and oxygen levels is likely to be important for the specific role of A-type proteins in the maturation of [4Fe-4S] cluster-containing proteins under aerobic growth or oxidative stress conditions. Only the [4Fe-4S]2+-NifIscA was competent for rapid activation of apo-nitrogenase Fe protein under anaerobic conditions. Apo-NifIscA was shown to accept clusters from [4Fe-4S] cluster-bound NifU via rapid intact cluster transfer, indicating a potential role as a cluster carrier for delivery of clusters assembled on NifU. Overall the results support the proposal that A-type proteins can function as carrier proteins for clusters assembled on U-type proteins and suggest that they are likely to supply [2Fe-2S] clusters rather than [4Fe-4S] for the maturation of [4Fe-4S] cluster-containing proteins under aerobic or oxidative stress growth conditions.
Vascular adhesion molecules regulate the migration of leukocytes from the blood into tissue during inflammation. Leukocyte binding to vascular cell adhesion molecule-1 (VCAM-1) activates signals in endothelial cells, including Rac1 and calcium fluxes. These VCAM-1 signals are required for leukocyte transendothelial migration on VCAM-1. However, it has not been reported whether the cytoplasmic domain of VCAM-1 is necessary for these signals. Interestingly, the 19 amino acid sequence of the VCAM-1 cytoplasmic domain is 100% conserved among many mammalian species, suggesting an important functional role for the domain. To examine the function of the VCAM-1 cytoplasmic domain, we deleted the VCAM-1 cytoplasmic domain or mutated the cytoplasmic domain at amino acids N724, S728, Y729, S730 or S737. The cytoplasmic domain and S728, Y729, S730 or S737 were necessary for leukocyte transendothelial migration. The S728 and Y729, but not S730 or S737, were necessary for VCAM-1 activation of calcium fluxes. In contrast, the S730 and S737, but not S728 or Y729 were necessary for VCAM-1 activation of Rac1. These functional data are consistent with our computational model of the structure of the VCAM-1 cytoplasmic domain as an alpha helix with S728/Y729 and S730/S737 on opposite sides of the alpha helix. Together, these data indicate that S728/Y729 and S730/S737 are distinct functional sites that coordinate VCAM-1 activation of calcium fluxes and Rac1 during leukocyte transendothelial migration.
VCAM-1; cytoplasmic domain; computational model; endothelial; Rac1; calcium; leukocyte migration
KaiA protein that stimulates KaiC phosphorylation in the cyanobacterial circadian clock was recently shown to be destabilized by dibromothymoquinone (DBMIB), thus revealing KaiA as a sensor of the plastoquinone (PQ) redox state and suggesting an indirect control of the clock by light through PQ redox changes. Here we show using X-ray crystallography that several DBMIBs are bound to KaiA dimer. Some binding modes are consistent with oligomerization of N-terminal KaiA pseudoreceiver domains and/or reduced inter-domain flexibility. DBMIB bound to the C-terminal KaiA (C-KaiA) domain and limited stimulation of KaiC kinase activity by C-KaiA in the presence of DBMIB demonstrate that the cofactor may weakly inhibit KaiA-KaiC binding.
Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E•FPP• Ca1a2X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors.
FPP; substrate analogue; FTase; protein prenylation; enzymology
Mutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation constant (Ka) of acetyl CoA, with the increase being greater for the Arg472 mutants. These results indicate that while both these residues are involved in the binding of acetyl CoA to the enzyme, Arg472 is more important than Arg427. The mutations had substantially smaller effects on the kcat for pyruvate carboxylation. Part of the effects of the mutations was to increase the Km for MgATP and the Ka for activation by free Mg2+ determined at saturating acetyl CoA concentrations. The inhibitory effects of the mutations on the rates of the enzyme-catalysed bicarbonate-dependent ATP cleavage, carboxylation of biotin and phosphorylation of ADP by carbamoyl phosphate indicate that the major locus of the effects of the mutations was in the biotin carboxylase (BC) domain active site. Even though both Arg427 and Arg472 are distant from the BC domain active site, it is proposed that their contacts with other residues in the allosteric domain, either directly or through acetyl CoA, affect the positioning and orientation of the biotin-carboxyl carrier protein (BCCP) domain and thus the binding of biotin at the BC domain active site. Based on the kinetic analysis proposed here it is proposed that mutations of Arg427 and Arg472 perturb these contacts and consequently the binding of biotin at the BC domain active site. Inhibition of pyruvate carboxylation by the allosteric inhibitor, L-aspartate, was largely unaffected by the mutation of either Arg427 or Arg472.
pyruvate carboxylase; allosteric regulator; site-directed mutagenesis; steady-state kinetics; sedimentation velocity
A number of histone-binding domains are implicated in cancer through improper binding of chromatin. In a clinically reported case of acute myeloid leukemia (AML), a genetic fusion protein between nucleoporin 98 and the third plant homeodomain (PHD) finger of JARID1A drives an oncogenic transcriptional program that is dependent on histone binding by the PHD finger. By exploiting the requirement for chromatin binding in oncogenesis, therapeutics targeting histone readers may represent a new paradigm in drug development. In this study, we developed a novel small molecule screening strategy that utilizes HaloTag technology to identify several small molecules that disrupt binding of the JARID1A PHD finger to histone peptides. Small molecule inhibitors were validated biochemically through affinity pull downs, fluorescence polarization, and histone reader specificity studies. One compound was modified through medicinal chemistry to improve its potency while retaining histone reader selectivity. Molecular modeling and site-directed mutagenesis of JARID1A PHD3 provided insights into the biochemical basis of competitive inhibition.
The BK polyoma virus is a leading cause of chronic post kidney transplantation rejection. One target for therapeutic intervention is the initial association of the BK virus with the host cell. We hypothesize that the rate of BKV infection can be curbed by competitively preventing viral binding to cells. The x-ray structures of homologous viruses complexed with N-terminal glycoproteins suggest that the BC and HI loops of the viral coat are determinant for binding and thereby, infection of the host cell. The large size of the viral coat precludes it from common biophysical and small molecule screening studies. Hence, we sought to develop a smaller protein template incorporating the identified binding loops of the BK viral coat in a manner that adequately mimics the binding activity of the BK virus coat protein to cells. Such a mimic may serve as a tool for the identification of inhibitors of BK viral progression. Herein, we report the design and characterization of a reduced-size and soluble template derived from a four helix protein—TM1526 of Thermatoga maritima archaea bacteria—which maintains the topological display of the BC and HI loops as found in the viral coat protein, VP1, of BKV. We demonstrate that the GT1b and GD1b sialogangliosides, which bind to the VP1 of BKV, also associate with our BKV-template. Employing a GFP-tagged template, we show host cell association that is dose dependent and that can be reduced by neuraminidase treatment. These data demonstrate that the BKV-template mimics the host-cell binding observed for the wild-type virus coat protein, VP1.
BK virus; viral coat protein; sialoganglioside receptors; polyoma viruses
The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp co-expression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression, and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp co-expression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.
protein aggregation; molecular chaperone; protein folding; scFv; periplasm
The P1B-type ATPases are a ubiquitous family of P-type ATPases involved in the transport of transition metal ions. Divided into subclasses on the basis of sequence characteristics and substrate specificity, these integral membrane transporters play key roles in metal homeostasis, metal tolerance, and the biosynthesis of metalloproteins. The P1B-4-ATPases have the simplest architecture of the five P1B-ATPase families and have been suggested to play a role in Co2+ transport. A P1B-4-ATPase from Sulfitobacter sp. NAS-14.1, designated sCoaT, has been cloned, expressed, and purified. Activity assays indicate that sCoaT is specific for Co2+. A single Co2+ binding site is present, and optical, electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectroscopic data are consistent with tetrahedral coordination by oxygen and nitrogen ligands, including a histidine and likely a water. Surprisingly, there is no evidence for coordination by sulfur. Mutation of a conserved cysteine residue, Cys 327, in the signature transmembrane SPC metal binding motif does not abolish ATP hydrolysis activity or affect the spectroscopic analysis, establishing that this residue is not involved in the initial Co2+ binding by sCoaT. In contrast, replacements of conserved transmembrane residues Ser 325, His 657, Glu 658, and Thr 661 with alanine abolish ATP hydrolysis activity and Co2+ binding, indicating that these residues are necessary for Co2+ transport. These data represent the first in vitro characterization of a P1B-4-ATPase and its Co2+ binding site.
Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1′ atom of ThDP (replaced by Val51), or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pKa of 6.2) for TSA formation from pH 7.0-7.7 in wild type GCL. This pKa is similar to the pKa of 6.1 for the [1′,4′-iminopyrimidine (IP)]/[4′-aminopyrimidinium (APH+)] protonic equilibrium, suggesting that the same group(s) control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as acetyl donor and glyoxylate as acceptor, implying that this substitutent favored pyruvate as donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the pre-decarboxylation intermediate analogs derived from acetylphosphinate, propionylphosphinate and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH+ form is accessible.
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3′→5′ mRNA decay. Some previous studies suggested that both m7GpppG and m7GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m7GDP (7-methylguanosine diphosphate) and m32,2,7GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5′→3′ mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode and yeast). Furthermore, whereas mononucleoside diphosphates (m7GDP and m32,2,7GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m7GTP and m32,2,7GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m7GTP and m32,2,7GTP are cleaved at a slower rate than their corresponding dinucleotides (m7GpppG and m32,2,7GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m7GDP, they have a high binding affinity for m7GDP and m7GDP potently inhibits DcpS hydrolysis of m7GpppG, suggesting that m7GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m7GDP in mRNA metabolic pathways, due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.