ABSTRACT
Serine ß-lactamases inactivate ß-lactam antibiotics in a two-step mechanism comprising acylation and deacylation. For the deacylation step, a water molecule is activated by a conserved glutamate residue to release the adduct from the enzyme. The third-generation cephalosporin ceftazidime is a poor substrate for the class A ß-lactamase BlaC from Mycobacterium tuberculosis but it can be hydrolyzed faster when the active site pocket is enlarged, as was reported for mutant BlaC P167S. The conformational change in the Ω-loop of the P167S mutant displaces the conserved glutamate (Glu166), suggesting it is not required for deacylation of the ceftazidime adduct. Here, we report the characterization of wild type BlaC and BlaC E166A at various pH values. The presence of Glu166 strongly enhances activity against nitrocefin but not ceftazidime, indicating it is indeed not required for deacylation of the adduct of the latter substrate. At high pH wild type BlaC was found to exist in two states, one of which converts ceftazidime much faster, resembling the open state previously reported for the BlaC mutant P167S. The pH-dependent switch between the closed and open states is caused by the loss at high pH of a low-barrier hydrogen bond, a proton shared between Asp172 and Asp179. These results illustrate how readily shifts in substrate specificity can occur as a consequence of subtle changes in protein structure.
ABSTRACT
This study investigated age-related radiographic changes in the distal parts of the forelimbs by radiographic evaluation and identified the radiographic changes associated with diseases specific to foals. The hoof angle (HA), distal phalanx angle (P3A), distal phalanx palmer angle (P3PA), distal interphalangeal joint angle (DIPJA), and metacarpophalangeal joint angle (MPJA) on lateromedial radiographs of forelimbs were measured on the day after birth (Day 1); at 1, 2, 4, 6, and 8 weeks of age; and then at monthly intervals until 12 months of age. HA and P3A significantly increased from 1 day to 4 weeks and 4 weeks to 3 months of age. The P3PA increased dramatically from 1 day to 1 week, 1 week to 2 weeks, and 2 weeks to 8 weeks of age, and then decreased after 3 months of age. DIPJA significantly decreased from 1 day to 2 weeks of age before increasing from 3 to 5 months of age. MPJA increased with age until 4 weeks, slightly decreased from 2 to 4 months of age, and then gradually decreased from 4 to 6 months of age. The findings indicate that foals' forelimbs typically show flexion of the fetlock and a broken backward hoof-pastern axis just after birth, an upright fetlock until 4 months of age, and a change to a mature conformation after 6 months of age. Physiological variants were correlated with the occurrence of common foal diseases during the radiographic evaluation periods.
Subject(s)
Hoof and Claw , Horses , Animals , Hoof and Claw/diagnostic imaging , Forelimb/diagnostic imaging , Radiography , Extremities , Bone and BonesABSTRACT
Organic ferroelectrics have received a great deal of interest due to their exclusive properties. However, organic ferroelectrics have not been fully explored, which hinders their practical application. Here, we presented a novel metal-free organic molecular ferroelectric [4-MCHA][ClO4 ] (1) (4-MCHA=trans-4-methylcyclohexylamine), which exhibits an above-room-temperature of 328â K. Strikingly, the single crystal structure analysis of 1 shows that the driving force of phase transition is related to the interesting chair-boat conformation change of 4-MCHA cation, in addition to the order-disorder transition of ClO4 - anion. Using piezoelectric response force microscopy (PFM), the presence of domains and the implemented polarization switching were clearly observed, which explicitly determined the presence of room-temperature ferroelectricity of 1. As far as we know, the ferroelectric phase transition mechanism attributed to the conformational change in a trans isomeric cation is very rare. This research enriched the path of designing ferroelectric materials and smart materials.
ABSTRACT
In the attempt to create a delivery system for an alkali-cation stimulated drug release, a computational study was conducted, aiming for the evaluation of synthetic access towards glycolipid crown ethers analogs and their potential for coordination-induced changes of packing constraints for molecular assemblies. The results disfavor amide-linkages for the creation of macrocycles around the inter-glycosidic bond of a disaccharide. Conformational changes upon cation coordination of the macrocycle decrease the intersection area for easily accessible macrocycles based on lactose. This leads to shrinking intersection areas upon alkali complexation. Maltose-based analogs, on the other hand, exhibited the targeted increase of the glycolipid intersection area and, hence, may be considered as a promising resource.
Subject(s)
Disaccharides , Lactose , Alkalies , Cations , GlycolipidsABSTRACT
Electrochemical aptasensors have been extensively used to quantify food contaminants (e.g., mycotoxin) by using high-affinity aptamer for target recognition. Yet, analytical performance of aptasensors using different aptamers can be varied for the same target. Here, four aptamers with different sequences (i.e., A22, A34, A42, and A45) of patulin (PAT) were selected to estimate sensing behaviors at electrodes with electrochemical (EC) and photoelectrochemical (PEC) assays. Synergistic effect of steric hindrance and electron transfer distance was found to significantly affect EC and PEC response for PAT at aptasensors fabricated with A22, A34, A42, or A45. Eventually, A22 emerged to be the optimal aptamer for aptasensing, despite the highest affinity of A42 to PAT. The A22-based EC-PEC dual-mode ratiometric aptasensor offered a linear range of 50 fg mL-1 - 500 ng mL-1 with a detection limit of 30 fg mL-1 for PAT, and it was applied to apple product (i.e., juice, puree) analysis.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Malus , Patulin , Patulin/analysis , Electrodes , Limit of Detection , Electrochemical Techniques , GoldABSTRACT
The activity of deubiquitinases (DUBs) is tightly regulated in eukaryotes via various mechanisms. One of the regulatory strategies is substrate-induced catalytic triad rearrangement, where ubiquitin-binding helps the DUB adopt an active conformation for catalysis. The crystal structure of the apo form of such a DUB, when not bound to ubiquitin, reveals an inactive conformation of the catalytic residues, necessitating the structure of the ubiquitin-bound form to visualize the active state of the DUB. Comparing the apo and ubiquitin-bound structures reveals conformational changes leading to catalytic activation. To capture the deubiquitinase in its ubiquitin-bound form, a series of activity-based ubiquitin probes (Ub-ABPs) harboring C-terminal electrophiles were designed to react with the catalytic nucleophile of cysteine protease DUBs. The resulting covalently linked DUB-ubiquitin complex is amendable for structural studies to probe the DUB-ubiquitin interface and the potential conformational change of the DUB. Here, we present a detailed protocol for the generation and purification of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) in complex with a Ub-ABP, ubiquitin-vinyl methyl ester (UbVME), and the subsequent structural analysis to characterize the catalytic state of the DUB.
Subject(s)
Deubiquitinating Enzymes , Ubiquitin , Ubiquitin/metabolism , Protein Binding , Catalysis , Deubiquitinating Enzymes/metabolismABSTRACT
Both metal ions and lipid membranes have a wide distribution in amyloid plaques and play significant roles in AD pathogenesis. Although influences of different metal ions or lipid vesicles on the aggregation of Aß peptides have been extensively studied, their combined effects are less understood. In this study, we reported a unique effect of copper ion on Aß aggregation in the presence of lipid vesicles, different from other divalent metal ions. Cu2+ in a super stoichiometric amount leads to the rapid formation of ß-sheet rich structure, containing abundant low molecular weight (LMW) oligomers. We demonstrated that oligomerization of Aß40 induced by Cu2+ binding was an essential prerequisite for the rapid conformation transition. Overall, the finding provided a new view on the complex triple system of Aß, copper ion and lipid vesicles, which might help understanding of Aß pathologies.
Subject(s)
Amyloid beta-Peptides , Copper , Copper/chemistry , Amyloid beta-Peptides/metabolism , Metals , Ions , LipidsABSTRACT
It has been revealed that slightly acid electrolysed water (SAEW) could delay enzymatic browning and melanin formation in food. In this work, multi-spectroscopic methods and UHPLC-Q-TOF-MS were combined to study the underlying reason. The reversible mixed-type inhibition mode of HOCl (main components in SAEW) was determined. The ground state complex formation quenched the intrinsic fluorescence of polyphenol oxidase (PPO) and it was stable at lower temperature. The PPO conformational change (transformation from α-helix to ß-sheet) induced by SAEW was confirmed by 3D fluorescence and Circular dichroism (CD) spectrum. Moreover, the driving force of the interaction between HOCl and PPO was hydrogen bond, which was validated by the molecular docking result. Besides, the formation of melanin related compounds including dihydroxyphenylalanine (DOPA), dopaquinone, dopachrome, 5,6-dihydroxyindole-2-carboxylic acid (DHICA), 5,6-dihydroxyindole (DHI), and 5,6-indolequinone were significantly inhibited by SAEW treatment. These results demonstrated the potential of SAEW as a PPO inhibitor in the food industry.
Subject(s)
Agaricales , Melanosis , Catechol Oxidase/chemistry , Melanins , Water , Molecular Docking SimulationABSTRACT
From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.
Subject(s)
Amines , Electron-Transferring Flavoproteins , Electron-Transferring Flavoproteins/metabolism , Oxidation-Reduction , Electron Transport , Flavin-Adenine Dinucleotide/metabolismABSTRACT
Umami taste receptor type 1 member 1/3 (T1R1/T1R3) heterodimer has multiple ligand-binding sites, most of which are located in T1R1-Venus flytrap domain (T1R1-VFT). However, the critical binding process of T1R1-VFT/umami ligands remains largely unknown. Herein, T1R1-VFT was prepared with a sufficient amount and functional activity, and its binding characteristics with typical umami molecules (monosodium l-glutamate, disodium succinate, beefy meaty peptide, and inosine-5'-monophosphate) were explored via multispectroscopic techniques and molecular dynamics simulation. The results showed that, driven mainly by hydrogen bond, van der Waals forces, and electrostatic interactions, T1R1-VFT bound to umami compound at 1:1 (stoichiometric interaction) and formed T1R1-VFT/ligand complex (static fluorescence quenching) with a weak binding affinity (Ka values: 252 ± 19 to 1169 ± 112 M-1). The binding process was spontaneous and exothermic (ΔG, -17.72 to -14.26 kJ mol-1; ΔH, -23.86 to -12.11 kJ mol-1) and induced conformational changes of T1R1-VFT, which was mainly reflected in slight unfolding of α-helix (Δα-helix < 0) and polypeptide chain backbone structure. Meanwhile, the binding of the four ligands stabilized the active conformation of the T1R1-VFT pocket. This work provides insight into the binding interaction between T1R1-VFT/umami ligands and improves understanding of how umami receptor recognizes specific ligand molecules.
Subject(s)
Droseraceae , Receptors, G-Protein-Coupled , Droseraceae/metabolism , Inosine , Ligands , Peptides/chemistry , Receptors, G-Protein-Coupled/metabolism , Sodium Glutamate , Succinates , TasteABSTRACT
The human VPS10 domain-containing receptor SorCS3 belongs to the Vps10p-domain receptor family and is an important receptor for regulating normal cellular functions via protein sorting. Here, we determined the cryo-EM structure of the full-length SorCS3 receptor and further found that there were at least three distinct conformations (monomer, M-shaped dimer and N-shaped dimer) of SorCS3 in the apo state. The differences between the two dimer conformations were caused by PKD1-2 assembly. In contrast to its homologous proteins, the conserved residues GLN198, ARG678, TYR430, GLU1020 and ASP1024 may be key points for its dimerization and for protein/polypeptide binding. These results showed the structural details of apo-SorCS3, which provides a foundation for elucidating the mechanism of protein sorting.
Subject(s)
Carrier Proteins , Nerve Tissue Proteins , Carrier Proteins/metabolism , Cryoelectron Microscopy , Humans , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Transport , Receptors, Cell Surface/metabolismABSTRACT
Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.
Subject(s)
Immediate-Early Proteins , Monomeric GTP-Binding Proteins , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-akt , Rapamycin-Insensitive Companion of mTOR Protein , Humans , Immediate-Early Proteins/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Monomeric GTP-Binding Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Sirolimus/pharmacology , Transcription Factors/metabolismABSTRACT
IGF1R plays an important role in regulating cellular metabolism and growth. As a single transmembrane protein, its structure is flexible. Although previous studies revealed some structures of IGF1R, the cryo-EM apo structures of the receptor have never been reported. Herein, we reported four distinct cryo-EM structures that reveal the apo states of IGF1R. These conformations were classified as "Resting states" and "Active states", according to the orientation of α-CT helices and structural symmetry. In addition, a "Ligand-pocket" was formed in the active conformations, which presented a new view of conformational changes of apo-IGF1R. These results suggest a new dynamic change model to show the details of why and how ligands can bind to IGF1R.
Subject(s)
Receptor, IGF Type 1 , Cryoelectron Microscopy/methods , Humans , Ligands , Protein Conformation , Receptor, IGF Type 1/chemistry , Receptor, IGF Type 1/ultrastructureABSTRACT
Protein's magic function stems from its structure and various analytical techniques have been developed for it. Among proteins, membrane proteins are encoded 20-30% of genomes, whereas cause challenges for many analytical techniques. For example, lots of membrane proteins cannot form single crystal structure required by X-ray crystallography. As for NMR, the measurements were hindered by the low tumbling rates of membrane (i.e., phospholipid bilayers) where membrane proteins exist. In addition, membrane proteins usually lay parallel to the surface of phospholipid bilayers or form transmembrane structure. No matter parallel or perpendicular to phospholipid bilayers surface, membrane proteins form monolayer structure which is also difficult for X-ray and NMR to provide high-resolution results. Because NMR and X-ray crystallography are the two major analytical techniques to address protein's structure, membrane proteins only contribute 2.4% to the solved protein databank. Surface FT-IR techniques can evaluate the conformation and orientation of membrane proteins by amide I band. Specifically for α-helical peptides/proteins, the orientation of the axis is critical to decide whether proteins form transmembrane structure. Notice that the traditional FT-IR can only provide "low-resolution" results. Here, 13C isotope was introduced into the nonamyloid component (NAC), which spans residues 61-95 of α-synuclein (α-syn). Then, p-polarized multiple-angle incidence resolution spectrometry (pMAIRS) was used to determine the orientation of a specific residue of α-helical NAC in monolayer. In general, pMAIRS is a novel technique to work complementary with X-ray and NMR to address membrane peptides/proteins structure with high resolution even in monolayer.
Subject(s)
Lipid Bilayers , alpha-Synuclein , Incidence , Lipid Bilayers/chemistry , Membrane Proteins , Peptides/chemistry , Phospholipids , Protein Conformation , Spectroscopy, Fourier Transform Infrared/methods , alpha-Synuclein/chemistryABSTRACT
Three isomeric derivatives of 2,2',2â³-(1,3,5-benzinetriyl)-tris(1-phenyl-1-H-benzimidazole) (TPBi) bearing ethyl groups on the N-phenyl moieties were synthesized to elucidate the effects of intramolecular interactions on spontaneous orientation polarization (SOP) in thin films. The films of the TPBi derivatives displayed enhanced SOP with a surface potential change of up to 1.8 times that for TPBi, and the p-substituted derivative exhibited the largest potential change reported to date (+141.0 mV/nm). Density functional theory calculations and single-crystal structure analysis suggest that the introduction of the ethyl groups switched the stable molecular conformation from C1 to C3 symmetry. Through analysis of the structural anisotropy in the films by spectral ellipsometry and two-dimensional (2D) grazing-incidence wide-angle X-ray scattering, we conclude that the conformational change of the molecules was the major factor underlying the SOP enhancement.
ABSTRACT
Type 1 insulin-like growth factor receptor (IGF1R) plays an important role in regulating cellular metabolism and cell growth and has been identified as an anticancer drug target. Although previous studies have revealed some structures of IGF1R with different ligands, the continuous dynamic conformation change remains unclear. Here, we report 10 distinct structures (7.9-3.6 Å) of IGF1R bound to IGF1 or insulin to reveal the polymorphic conformations of ligand-bound IGF1R. These results showed that the α-CT2, disulfide bond (C670-C670'), and FnIII-2 domains had the most flexible orientations for the conformational change that occurs when ligands bind to the receptor. In addition, we found one special conformation (tentatively named the diverter-switch state) in both complexes, which may be one of the apo-IGF1R forms under ligand-treatment conditions. Hence, these results illustrated the mechanism of how different ligands could bind to human IGF1R and provided a rational template for drug design.
Subject(s)
Insulin , Receptor, IGF Type 1 , Cell Proliferation , Cryoelectron Microscopy , Humans , Insulin/metabolism , Ligands , Protein Domains , Receptor, IGF Type 1/chemistry , Receptor, IGF Type 1/metabolismABSTRACT
Controllable regulations on the enzyme conformation to optimize catalytic performance are highly desired for the immobilized biocatalysts yet remain challenging. Covalent organic frameworks (COFs) possess confined channels with finely tunable pore environment, offering a promising platform for enzyme encapsulation. Herein, we covalently immobilized the cytochrome c (Cyt c) in the size-matched channels of COFs with different contents of anchoring site, and significant enhancement of the stability and activity (≈600 % relative activity compared with free enzyme) can be realized by optimizing the covalent interactions. Structural analyses on the immobilized Cyt c suggest that covalent bonding could induce conformational perturbation resulting in more accessible active sites. The effectiveness of the covalent interaction modulation together with the tailorable confined channels of COFs offers promise to develop high-performance biocatalysts.
Subject(s)
Metal-Organic Frameworks , Catalysis , Catalytic Domain , Enzymes, Immobilized/chemistry , Metal-Organic Frameworks/chemistryABSTRACT
Sorting-related receptor with A-type repeats (SORLA) is an important receptor for regulating normal cellular functions via protein sorting. Here, we determined the structures of the full-length SORLA and identified two distinct conformations of apo-SORLA using single-particle cryogenic electron microscopy. In contrast to homologous proteins, both monomer and dimer forms of SORLA existed in a neutral solution. Only three hydrogen bonds in the vicinity of the dimer interface implied the involvement in dimerization. The orientation of residue R490 was a key point for ligand binding. These results suggest a unique mechanism of SORLA dimerization for protein trafficking.
Subject(s)
Adaptor Proteins, Vesicular Transport , LDL-Receptor Related Proteins , Adaptor Proteins, Vesicular Transport/metabolism , Carrier Proteins/metabolism , Cryoelectron Microscopy , LDL-Receptor Related Proteins/metabolism , Protein TransportABSTRACT
Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.
Subject(s)
Transferrin/ultrastructure , Binding Sites/physiology , Crystallography, X-Ray/methods , Glycosylation , Humans , Hydrogen-Ion Concentration , Iron/metabolism , Models, Molecular , Protein Binding/physiology , Protein Conformation , Scattering, Small Angle , Serum/chemistry , Serum/metabolism , Transferrin/metabolism , X-Ray DiffractionABSTRACT
Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.