ABSTRACT
Protein structures calculated using NMR data are less accurate and less well-defined than they could be. Here we use the program ANSURR to show that this deficiency is at least in part due to a lack of hydrogen bond restraints. We describe a protocol to introduce hydrogen bond restraints into the structure calculation of the SH2 domain from SH2B1 in a systematic and transparent way and show that the structures generated are more accurate and better defined as a result. We also show that ANSURR can be used as a guide to know when the structure calculation is good enough to stop.
Subject(s)
src Homology Domains , Protein Conformation , Hydrogen Bonding , Models, Molecular , Magnetic Resonance SpectroscopyABSTRACT
In prion replication, the cellular form of prion protein (PrPC) must undergo a full conformational transition to its disease-associated fibrillar form. Transmembrane forms of PrP have been implicated in this structural conversion. The cooperative unfolding of a structural core in PrPC presents a substantial energy barrier to prion formation, with membrane insertion and detachment of parts of PrP presenting a plausible route to its reduction. Here, we examined the removal of residues 119-136 of PrP, a region which includes the first ß-strand and a substantial portion of the conserved hydrophobic region of PrP, a region which associates with the ER membrane, on the structure, stability and self-association of the folded domain of PrPC. We see an "open" native-like conformer with increased solvent exposure which fibrilises more readily than the native state. These data suggest a stepwise folding transition, which is initiated by the conformational switch to this "open" form of PrPC.
Subject(s)
Prion Proteins , Humans , Prion Proteins/chemistry , Prion Proteins/genetics , Protein Conformation, beta-Strand , Protein FoldingABSTRACT
Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence.
Subject(s)
Enterococcus faecalis , N-Acetylmuramoyl-L-alanine Amidase , Bacterial Proteins/metabolism , Cell Wall/chemistry , Cell Wall/metabolism , Enterococcus faecalis/chemistry , Enterococcus faecalis/metabolism , Ligands , N-Acetylmuramoyl-L-alanine Amidase/analysis , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Nuclear Magnetic Resonance, Biomolecular , Peptidoglycan/analysis , Peptidoglycan/chemistry , Peptidoglycan/metabolismABSTRACT
Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use ß-phosphoglucomutase (ßPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. ßPGM catalyzes the isomerization of ß-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. ßPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported ß-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.
ABSTRACT
Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced â¼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.
Subject(s)
Escherichia coli , Metabolic Engineering , Escherichia coli/metabolism , Fermentation , Metabolic Engineering/methods , Phenylalanine/genetics , Phenylalanine/metabolism , Styrene/metabolismABSTRACT
Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding (Kdâ¼300â¯nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics.
ABSTRACT
Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.
Subject(s)
Phosphotransferases (Phosphomutases)/metabolism , Protein Processing, Post-Translational , Allosteric Regulation , Allosteric Site , Crystallography, X-Ray , Enzyme Assays , Glucose-6-Phosphate/analogs & derivatives , Glucose-6-Phosphate/metabolism , Glucosephosphates/metabolism , Glycolysis , Isomerism , Kinetics , Molecular Conformation , Phosphorylation , Phosphotransferases (Phosphomutases)/genetics , Phosphotransferases (Phosphomutases)/isolation & purification , Phosphotransferases (Phosphomutases)/ultrastructure , Proline/chemistry , Protein Domains , Proton Magnetic Resonance Spectroscopy , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructureABSTRACT
Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP ß-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the ß2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.
Subject(s)
Prion Diseases/genetics , Prion Proteins/genetics , Prions/genetics , Protein Conformation , Animals , Humans , Point Mutation/genetics , Prion Diseases/pathology , Prion Proteins/ultrastructure , Prions/ultrastructure , Protein Conformation, beta-Strand/geneticsABSTRACT
Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.
Subject(s)
Lysostaphin/chemistry , Peptidoglycan/chemistry , Staphylococcus aureus/chemistry , Bacteriolysis/drug effects , Biofilms , Cell Wall/chemistry , Chromatography, High Pressure Liquid , DNA Mutational Analysis , Glycine/chemistry , Ligands , Magnetic Resonance Spectroscopy , Mutagenesis, Site-Directed , Peptides/chemistry , Protein Binding , Protein Domains , Recombinant Proteins/chemistry , src Homology DomainsABSTRACT
ß-Phosphoglucomutase (ßPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of ß-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a ß-glucose 1,6-bisphosphate intermediate. Substrate-free ßPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into ßPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free ßPGMWT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report 1H, 15N and 13C backbone resonance assignments of the P146A variant (ßPGMP146A) in its substrate-free form, where the K145-A146 peptide bond adopts a trans conformation in contrast to all crystal structures of ßPGMWT, where the K145-P146 peptide bond is cis. In ßPGMP146A millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect ßPGM crystal structures, and a chemical shift comparison between substrate-free ßPGMP146A and ßPGMWT confirms that the solution conformations are very similar, except for the D137-A147 loop. Hence, the isomerisation state of the 145-146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12-T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114-S116), and the D137-A147 loop (V141-A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic MgII ion and the L44-L53 loop responsible for substrate discrimination.
Subject(s)
Lactococcus lactis/enzymology , Mutant Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular , Phosphoglucomutase/chemistry , Mutant Proteins/genetics , Phosphoglucomutase/geneticsABSTRACT
Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.
Subject(s)
Antigens, Surface/immunology , Enterococcus faecalis/pathogenicity , Gram-Positive Bacterial Infections/immunology , Gram-Positive Bacterial Infections/microbiology , Muramidase/immunology , Polysaccharides/immunology , Virulence , Animals , Antigens, Surface/genetics , Antigens, Surface/metabolism , Antimicrobial Cationic Peptides/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enterococcus faecalis/genetics , Enterococcus faecalis/immunology , Gram-Positive Bacterial Infections/metabolism , Muramidase/metabolism , Mutagenesis , Mutation , Polysaccharides/metabolism , Zebrafish/growth & development , Zebrafish/immunology , Zebrafish/microbiologyABSTRACT
Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.
Subject(s)
Flap Endonucleases/chemistry , Catalytic Domain , Cations, Divalent/chemistry , DNA/chemistry , DNA/metabolism , Flap Endonucleases/metabolism , Fluorescence Resonance Energy Transfer , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphates/chemistry , Protein Conformation , Protein Structure, Secondary , Substrate SpecificityABSTRACT
We have used NMR and computational methods to characterize the dynamics of the ribonuclease barnase over a wide range of timescales in free and inhibitor-bound states. Using temperature- and denaturant-dependent measurements of chemical shift, we show that barnase undergoes frequent and highly populated hinge bending. Using relaxation dispersion, we characterize a slower and less populated motion with a rate of 750 ± 200 s-1, involving residues around the lip of the active site, which occurs in both free and bound states and therefore suggests conformational selection. Normal mode calculations characterize correlated hinge bending motions on a very rapid timescale. These three measurements are combined with previous measurements and molecular dynamics calculations on barnase to characterize its dynamic landscape on timescales from picoseconds to milliseconds and length scales from 0.1 to 2.5 nm. We show that barnase has two different large-scale fluctuations: one on a timescale of 10-9-10-6 s that has no free energy barrier and is a hinge bending that is determined by the architecture of the protein; and one on a timescale of milliseconds (i.e., 750 s-1) that has a significant free energy barrier and starts from a partially hinge-bent conformation. These two motions can be described as hierarchical, in that the more highly populated faster motion provides a platform for the slower (less probable) motion. The implications are discussed. The use of temperature and denaturant is suggested as a simple and general way to characterize motions on the intermediate ns-µs timescale.
ABSTRACT
Mycobacterium tuberculosis causes pulmonary tuberculosis (TB) and claims ~1.8 million human lives per annum. Host nitric oxide (NO) is important in controlling TB infection. M. tuberculosis WhiB1 is a NO-responsive Wbl protein (actinobacterial iron-sulfur proteins first identified in the 1970s). Until now, the structure of a Wbl protein has not been available. Here a NMR structural model of WhiB1 reveals that Wbl proteins are four-helix bundles with a core of three α-helices held together by a [4Fe-4S] cluster. The iron-sulfur cluster is required for formation of a complex with the major sigma factor (σA) and reaction with NO disassembles this complex. The WhiB1 structure suggests that loss of the iron-sulfur cluster (by nitrosylation) permits positively charged residues in the C-terminal helix to engage in DNA binding, triggering a major reprogramming of gene expression that includes components of the virulence-critical ESX-1 secretion system.
Subject(s)
Bacterial Proteins/metabolism , DNA/metabolism , Iron-Sulfur Proteins/metabolism , Mycobacterium tuberculosis/metabolism , Nitric Oxide/metabolism , Transcription Factors/metabolism , Bacterial Proteins/chemistry , Gene Expression Regulation, Bacterial , Iron-Sulfur Proteins/chemistry , Magnetic Resonance Spectroscopy , Mycobacterium tuberculosis/chemistry , Protein Conformation, alpha-Helical , Protein Structure, Tertiary , Sigma Factor/metabolism , Transcription Factors/chemistry , Type VII Secretion Systems/geneticsABSTRACT
Formation of magnetite nanocrystals by magnetotactic bacteria is controlled by specific proteins which regulate the particles' nucleation and growth. One such protein is Mms6. This small, amphiphilic protein can self-assemble and bind ferric ions to aid in magnetite formation. To understand the role of Mms6 during in vitro iron oxide precipitation we have performed in situ pH titrations. We find Mms6 has little effect during ferric salt precipitation, but exerts greatest influence during the incorporation of ferrous ions and conversion of this salt to mixed-valence iron minerals, suggesting Mms6 has a hitherto unrecorded ferrous iron interacting property which promotes the formation of magnetite in ferrous-rich solutions. We show ferrous binding to the DEEVE motif within the C-terminal region of Mms6 by NMR spectroscopy, and model these binding events using molecular simulations. We conclude that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ions predominate.
ABSTRACT
Mutations in polycystin-1 (PC1) give rise to autosomal dominant polycystic kidney disease, an important and common cause of kidney failure. Despite its medical importance, the function of PC1 remains poorly understood. Here, we investigated the role of the intracellular polycystin-1, lipoxygenase, and α-toxin (PLAT) signature domain of PC1 using nuclear magnetic resonance, biochemical, cellular, and in vivo functional approaches. We found that the PLAT domain targets PC1 to the plasma membrane in polarized epithelial cells by a mechanism involving the selective binding of the PLAT domain to phosphatidylserine and L-α-phosphatidylinositol-4-phosphate (PI4P) enriched in the plasma membrane. This process is regulated by protein kinase A phosphorylation of the PLAT domain, which reduces PI4P binding and recruits ß-arrestins and the clathrin adaptor AP2 to trigger PC1 internalization. Our results reveal a physiological role for the PC1-PLAT domain in renal epithelial cells and suggest that phosphorylation-dependent internalization of PC1 is closely linked to its function in renal development and homeostasis.
Subject(s)
Lipoxygenase/physiology , TRPP Cation Channels/physiology , Humans , Lipoxygenase/genetics , Mutation , Protein Structure, Tertiary , TRPP Cation Channels/geneticsABSTRACT
The effects of sodium thiocyanate, sodium chloride, and sodium sulfate on the ribonuclease barnase were studied using differential scanning calorimetry (DSC) and NMR. Both measurements reveal specific and saturable binding at low anion concentrations (up to 250 mM), which produces localized conformational and energetic effects that are unrelated to the Hofmeister series. The binding of sulfate slows intramolecular motions, as revealed by peak broadening in 13C heteronuclear single quantum coherence spectroscopy. None of the anions shows significant binding to hydrophobic groups. Above 250 mM, the DSC results are consistent with the expected Hofmeister effects in that the chaotropic anion thiocyanate destabilizes barnase. In this higher concentration range, the anions have approximately linear effects on protein NMR chemical shifts, with no evidence for direct interaction of the anions with the protein surface. We conclude that the effects of the anions on barnase are mediated by solvent interactions. The results are not consistent with the predictions of the preferential interaction, preferential hydration, and excluded volume models commonly used to describe Hofmeister effects. Instead, they suggest that the Hofmeister anion effects on both stability and solubility of barnase are due to the way in which the protein interacts with water molecules, and in particular with water dipoles, which are more ordered around sulfate anions and less ordered around thiocyanate anions.
ABSTRACT
Polycystin-1 is a large transmembrane protein mutated in the common genetic disorder autosomal dominant polycystic kidney disease. One of the predicted intracellular domains of polycystin-1 is PLAT (Polycystin-1, Lipoxygenase and Alpha Toxin), which consists of 116 amino acids and is anchored to the membrane by linkers at both ends. It is predicted to have a large number of hydrophobic residues on the surface. Assignment of the NMR spectrum was hampered by considerable line broadening, and hence a programme of site-directed mutagenesis and searching for suitable solution conditions was undertaken. The optimum construct required fusion of the GB1 domain at the N-terminus and a His tag at the C-terminus, and proved to have several additional amino acids at both ends beyond the canonical domain boundaries, as well as mutation of W3128 to alanine. Optimum solubility required 500 mM sodium chloride, and usable spectra could only be obtained by perdeuteration. Backbone assignment was made using standard triple resonance spectra and is 88 % complete. The chemical shifts obtained suggest that a loop consisting of residues 3223-3228 is mobile in solution, and that the protein is similar in structure to a prediction produced by Swiss-Model based on the structure of a homologous protein.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , TRPP Cation Channels/chemistry , Humans , Models, Molecular , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM-peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Enterococcus faecalis/metabolism , Peptidoglycan/metabolism , Bacterial Proteins/genetics , Enterococcus faecalis/chemistry , Enterococcus faecalis/genetics , Peptidoglycan/chemistry , Protein Binding , Protein Structure, TertiaryABSTRACT
Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aß binding to g3p. NMR studies show that g3p binding to Aß fibers is predominantly through middle and C-terminal residues of the Aß subunit, indicating ß strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aß fibers (fAß) (KD=9.4nM); (2); blocks fAß assembly (IC50~50nM) and (3) dissociates fAß (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.