ABSTRACT
The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.
Subject(s)
Acyltransferases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Pyrazoles/pharmacology , Acyltransferases/metabolism , Anti-Bacterial Agents/metabolism , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Imidazoles/metabolism , Microbial Sensitivity Tests , Protein Binding , Pyrazoles/metabolismABSTRACT
Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the 'molecular brake', 'DFG latch', 'A-loop plug', and 'αC tether'. The first three sites repress the kinase from adopting an active conformation, whereas the αC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs.
Subject(s)
Allosteric Regulation , Receptor, Fibroblast Growth Factor, Type 2/chemistry , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Conformation , Receptor, Fibroblast Growth Factor, Type 2/geneticsABSTRACT
The design of folded miniature proteins is predicated on establishing noncovalent interactions that direct the self-assembly of discrete thermostable tertiary structures. In this work, we describe how a network of cation-π interactions present in proteins containing "WSXWS motifs" can be emulated to stabilize the core of a miniature protein. This 19-residue protein sequence recapitulates a set of interdigitated arginine and tryptophan residues that stabilize a distinctive ß-strand:loop:PPII-helix topology. Validation of the compact fold determined by NMR was carried out by mutagenesis of the cation-π network and by comparison to the corresponding disulfide-bridged structure. These results support the involvement of a coordinated set of cation-π interactions that stabilize the tertiary structure.
Subject(s)
Cations/chemistry , Proteins/chemistry , Amino Acid Motifs , Amino Acid Sequence , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G-protein-mediated signaling. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Under physiological conditions, the rhodopsin-arrestin-1 interaction occurs in highly specialized disk membrane in which rhodopsin resides. This membrane is replaced with mimetics when working with purified proteins. While detergents are commonly used as membrane mimetics, most detergents denature arrestin-1, preventing biochemical studies of this interaction. In contrast, bicelles provide a suitable alternative medium. An advantage of bicelles is that they contain lipids, which have been shown to be necessary for normal rhodopsin-arrestin-1 interaction. Here we describe how to reconstitute rhodopsin into bicelles, and how bicelle properties affect the rhodopsin-arrestin-1 interaction.
Subject(s)
Arrestin/chemistry , Arrestin/metabolism , Rhodopsin/chemistry , Rhodopsin/metabolism , Models, Biological , Phosphorylation , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiologyABSTRACT
Secondary active transporters undergo large conformational changes to facilitate the efflux of substrates across the lipid bilayer. Among the smallest known transport proteins are members of the small multidrug resistance (SMR) family that are composed of four transmembrane (TM) domains and assemble into dimers. An unanswered question in the SMR field is how the dimerization domain (TM4) is coupled with the substrate-binding chamber (TM1-3). To provide insight for this essential aspect of ion-coupled transport, we carried out a structure-function study on the SMR protein EmrE using solid-state NMR spectroscopy in lipid bilayers and resistance assays in Escherichia coli. The chemical shifts for EmrE were consistent with ß-strand secondary structure for the loop connecting TM3 and TM4. Based on these structural results, EmrE mutants were created to ascertain whether a specific loop length and composition were necessary for function. A linker encompassing six extra Gly residues relative to wild-type EmrE failed to give resistance; however, the number of residues in the loop was not the only criterion for a functional efflux pump. Replacement of the central hydrophobic residue with Gly (L83G) also conferred no ethidium resistance phenotype, which supported the conclusion that the structure and length of the loop were both essential for ion-coupled transport. Taken together with a bioinformatics analysis, a structured linker is likely conserved across the SMR family to play an active role in mediating the conformational switch between inward-open and outward-open states necessary for drug efflux. These findings underscore the important role loops can play in mediating efflux.
Subject(s)
Antiporters/chemistry , Drug Resistance, Multiple/genetics , Escherichia coli Proteins/chemistry , Lipid Bilayers/chemistry , Structure-Activity Relationship , Antiporters/genetics , Antiporters/ultrastructure , Binding Sites , Escherichia coli Proteins/genetics , Escherichia coli Proteins/ultrastructure , Lipid Bilayers/metabolism , Magnetic Resonance Spectroscopy , Protein Binding , Protein Conformation , Protein Multimerization , Protein Structure, Tertiary/genetics , Substrate SpecificityABSTRACT
EmrE is a multidrug resistance efflux pump with specificity to a wide range of antibiotics and antiseptics. To obtain atomic-scale insight into the attributes of the native state that encodes the broad specificity, we used a hybrid of solution and solid-state NMR methods in lipid bilayers and bicelles. Our results indicate that the native EmrE dimer oscillates between inward and outward facing structural conformations at an exchange rate (k(ex)) of ~300 s(-1) at 37 °C (millisecond motions), which is ~50-fold faster relative to the tetraphenylphosphonium (TPP(+)) substrate-bound form of the protein. These observables provide quantitative evidence that the rate-limiting step in the TPP(+) transport cycle is not the outward-inward conformational change in the absence of drug. In addition, using differential scanning calorimetry, we found that the width of the gel-to-liquid crystalline phase transition was 2 °C broader in the absence of the TPP(+) substrate versus its presence, which suggested that changes in transporter dynamics can impact the phase properties of the membrane. Interestingly, experiments with cross-linked EmrE showed that the millisecond inward-open to outward-open dynamics was not the culprit of the broadening. Instead, the calorimetry and NMR data supported the conclusion that faster time scale structural dynamics (nanosecond-microsecond) were the source and therefore impart the conformationally plastic character of native EmrE capable of binding structurally diverse substrates. These findings provide a clear example how differences in membrane protein transporter structural dynamics between drug-free and bound states can have a direct impact on the physical properties of the lipid bilayer in an allosteric fashion.
Subject(s)
Antiporters/chemistry , Antiporters/genetics , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Cross-Linking Reagents , Escherichia coli/chemistry , Escherichia coli/genetics , Indicators and Reagents , Lipid Bilayers , Magnetic Resonance Spectroscopy , Onium Compounds/chemistry , Organophosphorus Compounds/chemistry , Protein ConformationABSTRACT
Protein folding and unfolding are crucial for a range of biological phenomena and human diseases. Defining the structural properties of the involved transient species is therefore of prime interest. Using a combination of cold denaturation with NMR spectroscopy, we reveal detailed insight into the unfolding of the homodimeric repressor protein CylR2. Seven three-dimensional structures of CylR2 at temperatures from 25 °C to -16 °C reveal a progressive dissociation of the dimeric protein into a native-like monomeric intermediate followed by transition into a highly dynamic, partially folded state. The core of the partially folded state seems critical for biological function and misfolding.
Subject(s)
Bacterial Proteins/chemistry , Cytotoxins/chemistry , DNA, Bacterial/chemistry , Enterococcus faecalis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cold Temperature , Cytotoxins/genetics , Cytotoxins/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Enterococcus faecalis/genetics , Escherichia coli/genetics , Models, Molecular , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular , Promoter Regions, Genetic , Protein Binding , Protein Denaturation , Protein Folding , Protein Multimerization , Protein Unfolding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 1980s.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Proteins/chemistry , Animals , Drug Discovery , History, 20th Century , History, 21st Century , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular/history , Protein Conformation , Protein Interaction Mapping/history , Protein Interaction Mapping/methods , Proteins/metabolismABSTRACT
Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 µM), its affinity for P-Rh (K(D) ~80 µM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K(D) of~50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.
Subject(s)
Arrestin/chemistry , Arrestin/metabolism , Rhodopsin/metabolism , Arrestin/genetics , Binding Sites , Humans , Kinetics , Models, Molecular , Multiprotein Complexes/chemistry , Mutagenesis, Insertional , Nuclear Magnetic Resonance, Biomolecular , Opsins/chemistry , Opsins/metabolism , Phosphorylation , Photochemical Processes , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodopsin/chemistryABSTRACT
A rotavirus G1P[8] strain C1-81 was isolated from a 5-month-old female infant admitted to hospital with fever and severe diarrhea in Incheon, South Korea. To investigate its full genomic relatedness and its group, the full genome of strain C1-81 was determined. Based on a full genome classification system, C1-81 was shown to possess the typical Wa-like genotype constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. On the basis of sequence similarities, the strain was shown to be the closest related strain to contemporary human rotavirus strains with recent strains isolated in Asia. This C1-81 strain showed the highest degree of nucleic acid similarity (98.8% and 97%) to G1 B4633-03 and P[8] (Thai-1604 and Dhaka8-02), respectively. This is the first report that group A rotavirus was analyzed with G1P[8] in South Korea. The study of the complete genome of the virus will help understanding of the evolution of rotavirus.
Subject(s)
Genome, Viral , RNA, Viral/genetics , Rotavirus/genetics , Sequence Analysis, DNA , Diarrhea/virology , Female , Genotype , Humans , Infant , Molecular Sequence Data , Phylogeny , Republic of Korea , Rotavirus/isolation & purification , Rotavirus Infections/virology , Sequence Homology, Nucleic AcidABSTRACT
Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the ß-strand ß3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.
Subject(s)
Hydrophobic and Hydrophilic Interactions , Intramolecular Oxidoreductases/chemistry , Leucine/chemistry , Macrophage Migration-Inhibitory Factors/chemistry , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Molecular Sequence Data , Mutant Proteins/chemistry , Mutation/genetics , Protein Binding/genetics , Protein Multimerization , Protein Stability , Protein Structure, Quaternary , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
Conformational changes are essential for protein-protein and protein-ligand recognition. Here we probed changes in the structure of the protein ubiquitin at low temperatures in supercooled water using NMR spectroscopy. We demonstrate that ubiquitin is well folded down to 263 K, although slight rearrangements in the hydrophobic core occur. However, amide proton chemical shifts show non-linear temperature dependence in supercooled solution and backbone hydrogen bonds become weaker in the region that is most prone to cold-denaturation. Our data suggest that the weakening of the hydrogen bonds in the ß-sheet of ubiquitin might be one of the first events that occur during cold-denaturation of ubiquitin. Interestingly, the same region is strongly involved in ubiquitin-protein complexes suggesting that this part of ubiquitin more easily adjusts to conformational changes required for complex formation.
Subject(s)
Cold Temperature , Ubiquitin/chemistry , Amides/chemistry , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Ligands , Magnetic Resonance Spectroscopy/methods , Nonlinear Dynamics , Protein Binding , Protein Conformation , Protein Structure, Secondary , Temperature , Ubiquitin/physiology , Water/chemistryABSTRACT
The major component of neural inclusions that are the pathological hallmark of Parkinson's disease are amyloid fibrils of the protein α-synuclein (aS). Here we investigated if the disease-related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two-dimensional proton-detected high-resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of ß-strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano-second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation-induced change in the conformation of aS aggregates.
Subject(s)
Amyloid/chemistry , Amyloid/genetics , Mutation , alpha-Synuclein/chemistry , alpha-Synuclein/genetics , Amyloid/metabolism , Humans , Nuclear Magnetic Resonance, Biomolecular , alpha-Synuclein/metabolismABSTRACT
Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein-protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.
Subject(s)
Crystallography, X-Ray/methods , Electron Spin Resonance Spectroscopy/methods , Magnetic Resonance Spectroscopy/methods , Proteins/chemistry , Spin LabelsABSTRACT
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.
Subject(s)
Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Chemotaxis/drug effects , Glucocorticoids , Humans , Inhibitory Concentration 50 , Macrophage Migration-Inhibitory Factors/genetics , Mutation , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity RelationshipABSTRACT
Soluble oligomers are potent toxins in many neurodegenerative diseases, but little is known about the structure of soluble oligomers and their structure-toxicity relationship. Here we prepared on-pathway oligomers of the 140-residue protein alpha-synuclein, a key player in Parkinson's disease, at concentrations an order of magnitude higher than previously possible. The oligomers form ion channels with well-defined conductance states in a variety of membranes, and their beta-structure differs from that of amyloid fibrils of alpha-synuclein.
Subject(s)
Biopolymers/chemistry , alpha-Synuclein/chemistry , Blotting, Western , Microscopy, Atomic Force , Nuclear Magnetic Resonance, BiomolecularABSTRACT
The relation of alpha-synuclein (alphaS) aggregation to Parkinson's disease has long been recognized, but the pathogenic species and its molecular properties have yet to be identified. To obtain insight into the properties of alphaS in an aggregation-prone state, we studied the structural properties of alphaS at acidic pH using NMR spectroscopy and computation. NMR demonstrated that alphaS remains natively unfolded at lower pH, but secondary structure propensities were changed in proximity to acidic residues. The ensemble of conformations of alphaS at acidic pH is characterized by a rigidification and compaction of the Asp and Glu-rich C-terminal region, an increased probability for proximity between the NAC-region and the C-terminal region and a lower probability for interactions between the N- and C-terminal regions.
Subject(s)
alpha-Synuclein/chemistry , Amino Acid Sequence , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Parkinson Disease/metabolism , Protein Conformation , Protein FoldingABSTRACT
A synthetic cell-permeable peptide corresponding to the highly conserved alpha-integrin signature motif, Palmityl-K(989)VGFFKR(995) (Pal-FF), induces integrin activation and aggregation in human platelets. Systematic replacement of the F(992)-F(993) with amino acids of greater or lesser hydrophobicity to create Pal-KVGxxKR peptides demonstrate that hydrophobic amino acids (isoleucine, phenylalanine, tyrosine, tryptophan) are essential for agonist potency. In marked contrast, substitution with small and/or hydrophilic amino acids (glycine, alanine, serine) causes a switch in the biological activity resulting in inhibition of platelet aggregation, adhesion, ADP secretion, and thromboxane synthesis. These substituted, hydrophilic peptides are not true pharmacological antagonists, as they actively induce a phosphotyrosine signaling cascade in platelets. Singly substituted peptides (Pal-AF and Pal-FA) cause preferential retention of pro- or anti-thrombotic properties, respectively. Because the alpha-integrin signature motif is an established docking site for a number of diverse cytoplasmic proteins, we conclude that eliminating critical protein-protein interactions mediated through the hydrophobic amino acids, especially F(993), favors an anti-thrombotic pathway in platelets. Agents derived from the inhibitory peptides described in this study may represent a new therapeutic strategy for anti-platelet or anti-integrin drug development.