RÉSUMÉ
Inulin has found commercial applications in the pharmaceutical, nutraceutical, and food industries due to its beneficial health effects. The enzymatic biosynthesis of microbial inulin has garnered increasing attention. In this study, molecular modification was applied to Lactobacillus mulieris UMB7800 inulosucrase, an enzyme that specifically produces high-molecular weight inulin, to enhance its catalytic activity and thermostability. Among the 18 variable regions, R5 was identified as a crucial region significantly impacting enzymatic activity by replacing it with more conserved sequences. Site-directed mutagenesis combined with saturated mutagenesis revealed that the mutant A250 V increased activity by 68%. Additionally, after screening candidate mutants by rational design, four single-point mutants, S344D, H434P, E526D, and G531P, were shown to enhance thermostability. The final combinational mutant, M5, exhibited a 66% increase in activity and a 5-fold enhancement in half-life at 55 °C. These findings are significant for understanding the catalytic activity and thermostability of inulosucrase and are promising for the development of microbial inulin biosynthesis platforms.
Sujet(s)
Protéines bactériennes , Stabilité enzymatique , Hexosyltransferases , Inuline , Lactobacillus , Mutagenèse dirigée , Inuline/métabolisme , Inuline/composition chimique , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Protéines bactériennes/composition chimique , Hexosyltransferases/génétique , Hexosyltransferases/métabolisme , Hexosyltransferases/composition chimique , Lactobacillus/enzymologie , Lactobacillus/génétique , Lactobacillus/métabolisme , Cinétique , Température élevée , Ingénierie des protéines , Spécificité du substratRÉSUMÉ
The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.
Sujet(s)
Interleukine-6 , Simulation de docking moléculaire , Liaison aux protéines , Récepteurs à l'interleukine-6 , Interleukine-6/métabolisme , Interleukine-6/génétique , Humains , Récepteurs à l'interleukine-6/métabolisme , Récepteurs à l'interleukine-6/génétique , Récepteurs à l'interleukine-6/composition chimique , Récepteur gp130 de cytokines/métabolisme , Récepteur gp130 de cytokines/génétique , Récepteur gp130 de cytokines/composition chimique , Mutagenèse dirigée , Sites de fixation , Facteur de transcription STAT-3/métabolisme , Phosphorylation , MutationRÉSUMÉ
Haloalkane dehalogenase, LinB, is a member of the α/ß hydrolase family of enzymes. It has a wide range of halogenated substrates, but, has been mostly studied in context of degradation of hexachlorocyclohexane (HCH) isomers, especially ß-HCH (5-12% of total HCH isomers), which is the most recalcitrant and persistent among all the HCH isomers. LinB was identified to directly act on ß-HCH in a one or two step transformation which decreases its toxicity manifold. Thereafter, many studies focused on LinB including its structure determination using X-ray crystallographic studies, structure comparison with other haloalkane dehalogenases, substrate specificity and kinetic studies, protein engineering and site-directed mutagenesis studies in search of better catalytic activity of the enzyme. LinB was mainly identified and characterized in bacteria belonging to sphingomonads. Detailed sequence comparison of LinB from different sphingomonads further revealed the residues critical for its activity and ability to catalyze either one or two step transformation of ß-HCH. Association of LinB with IS6100 elements is also being discussed in detail in sphingomonads. In this review, we summarized vigorous efforts done by different research groups on LinB for developing better bioremediation strategies against HCH contamination. Also, kinetic studies, protein engineering and site directed mutagenesis studies discussed here forms the basis of further exploration of LinB's role as an efficient enzyme in bioremediation projects.
Sujet(s)
Lindane , Hydrolases , Hydrolases/métabolisme , Hydrolases/génétique , Hydrolases/composition chimique , Lindane/métabolisme , Spécificité du substrat , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/composition chimique , Cinétique , Dépollution biologique de l'environnement , Cristallographie aux rayons X , Mutagenèse dirigée , Sphingomonas/enzymologie , Sphingomonas/génétique , Sphingomonas/métabolismeRÉSUMÉ
Mogrosides are natural compounds highly valued in the food sector for their exceptional sweetness. Here, we report a novel O-glycosyltransferase (UGT74DD1) from Siraitia grosvenorii that catalyzes the conversion of mogrol to mogroside IIE. Site-directed mutagenesis yielded the UGT74DD1-W351A mutant, which exhibited the new capability to transform mogroside IIE into the valuable sweetener mogroside III, but with low catalytic activity. Subsequently, using structure-guided directed evolution with combinatorial active-site saturation testing, the superior mutant M6 (W351A/Q373 K/E49H/Q335W/S278C/D17F) were obtained, which showed a 46.1-fold increase in catalytic activity compared to UGT74DD1-W351A. Molecular dynamics simulations suggested that the enhanced activity and extended substrate profiles of M6 are due to its enlarged substrate-binding pocket and strengthened enzyme-substrate hydrogen bonding interactions. Overall, we redesigned UGT74DD1, yielding mutants that catalyze the conversion of mogrol into mogroside III. This study thus broadens the toolbox of UGTs capable of catalyzing the formation of valuable polyglycoside compounds.
Sujet(s)
Glycosyltransferase , Édulcorants , Glycosyltransferase/génétique , Glycosyltransferase/composition chimique , Glycosyltransferase/métabolisme , Édulcorants/composition chimique , Édulcorants/métabolisme , Cucurbitaceae/composition chimique , Cucurbitaceae/enzymologie , Cucurbitaceae/génétique , Cucurbitaceae/métabolisme , Mutagenèse dirigée , Protéines végétales/génétique , Protéines végétales/composition chimique , Protéines végétales/métabolisme , Biocatalyse , Domaine catalytique , Ingénierie des protéines , Spécificité du substrat , CinétiqueRÉSUMÉ
Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing ß1- versus ß2-adrenergic receptors (ß1AR and ß2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of ß2AR, but not ß1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on ß2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical ß2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR.
Sujet(s)
Microbiome gastro-intestinal , Glutamine , Myocytes cardiaques , Récepteurs bêta-2 adrénergiques , Animaux , Humains , Récepteurs bêta-2 adrénergiques/métabolisme , Récepteurs bêta-2 adrénergiques/génétique , Régulation allostérique , Souris , Mâle , Myocytes cardiaques/métabolisme , Myocytes cardiaques/effets des médicaments et des substances chimiques , Glutamine/métabolisme , Cellules HEK293 , Simulation de docking moléculaire , Défaillance cardiaque/métabolisme , Défaillance cardiaque/microbiologie , Mutagenèse dirigée , Récepteurs bêta-1 adrénergiques/métabolisme , Récepteurs bêta-1 adrénergiques/génétique , Souris de lignée C57BLRÉSUMÉ
The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.
Sujet(s)
Aryl hydrocarbon hydroxylases , Famille-2 de cytochromes P450 , Flavine mononucléotide , NADPH-ferrihemoprotéine reductase , NADPH-ferrihemoprotéine reductase/métabolisme , NADPH-ferrihemoprotéine reductase/composition chimique , NADPH-ferrihemoprotéine reductase/génétique , Flavine mononucléotide/métabolisme , Flavine mononucléotide/composition chimique , Famille-2 de cytochromes P450/métabolisme , Famille-2 de cytochromes P450/génétique , Famille-2 de cytochromes P450/composition chimique , Aryl hydrocarbon hydroxylases/composition chimique , Aryl hydrocarbon hydroxylases/métabolisme , Aryl hydrocarbon hydroxylases/génétique , Humains , Mutagenèse dirigée , Domaines protéiques , Cinétique , AnimauxRÉSUMÉ
(R)-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven (R)-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi, virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency (kcat/Km) than the wild-type enzyme. Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of (R)-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of (R)-3-aminobutanol.
Sujet(s)
Ingénierie des protéines , Transaminases , Transaminases/métabolisme , Transaminases/génétique , Transaminases/composition chimique , Ingénierie des protéines/méthodes , Spécificité du substrat , Sites de fixation , Biocatalyse , Mutagenèse , Mutagenèse dirigée , Modèles moléculaires , Burkholderiaceae/enzymologie , Burkholderiaceae/génétique , CinétiqueRÉSUMÉ
Mutational study is a cornerstone methodology in biochemistry and genetics, and many mutagenesis strategies have been invented to promote the efficiency of gene engineering. In this study, we developed a simple and timesaving approach to integrate simultaneous mutagenesis at discrete sites. By using plasmid as a template and compatible oligonucleotide primers per the QuikChange strategy, our method was able to introduce multiple nucleotide insertions, deletions and replacements in one round of polymerase chain reaction. The longest insertion and deletion were achieved with 28 bp and 16 bp mismatch respectively. For minor nucleotide replacements (mismatch no more than 4 bp), mutations were achieved at up to 4 discrete locations. Usually, a successful clone with all desired mutations was found by screening 5 colonies. Clones with a subset of mutations may be stocked into the library of mutants or used as templates in the next rounds of mutagenic PCR to accomplish the entire construction project. This method can be applied to build up a combinatory library of mutants through saturation mutagenesis at multiple sites. It is promising to facilitate the research of protein biochemistry, forward genetics and synthetic biology.
Sujet(s)
Mutagenèse dirigée , Plasmides , Réaction de polymérisation en chaîne , Plasmides/génétique , Réaction de polymérisation en chaîne/méthodes , Mutagenèse dirigée/méthodes , ADN/génétiqueRÉSUMÉ
Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH â¼ 4.7), at a pH â¼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.
Sujet(s)
ADN simple brin , Électro-osmose , Hémolysines , Mutagenèse dirigée , Nanopores , Concentration en ions d'hydrogène , ADN simple brin/composition chimique , ADN simple brin/génétique , Hémolysines/composition chimique , Hémolysines/génétique , Électricité statiqueRÉSUMÉ
The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.
Sujet(s)
D-amino-acid oxidase , Ingénierie des protéines , D-amino-acid oxidase/génétique , D-amino-acid oxidase/métabolisme , D-amino-acid oxidase/composition chimique , Ingénierie des protéines/méthodes , Spécificité du substrat , Mutagenèse , Mutagenèse dirigée/méthodes , Amino-butyrates/métabolisme , Modèles moléculaires , Mutation , Sites de fixationRÉSUMÉ
Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15N and 13C HSQC NMR spectroscopy to gain structural insight into the CuSO4-mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.
Sujet(s)
Cystéine , Galectine 1 , Oxydoréduction , Galectine 1/métabolisme , Galectine 1/composition chimique , Galectine 1/génétique , Humains , Cystéine/métabolisme , Cystéine/composition chimique , Disulfures/métabolisme , Disulfures/composition chimique , Pliage des protéines , Dépliement des protéines , Modèles moléculaires , Lactose/métabolisme , Lactose/composition chimique , Mutagenèse dirigéeRÉSUMÉ
Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.
Sujet(s)
Mutagenèse dirigée , Récepteur IGF de type 1 , Récepteur à l'insuline , Récepteur à l'insuline/génétique , Récepteur à l'insuline/composition chimique , Récepteur à l'insuline/métabolisme , Humains , Récepteur IGF de type 1/génétique , Récepteur IGF de type 1/composition chimique , Récepteur IGF de type 1/métabolisme , Multimérisation de protéines , , Antigènes CDRÉSUMÉ
Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.
Sujet(s)
Canaux ioniques sensibles à l'acidité , Encéphalopathie ischémique , Acide glutamique , Animaux , Femelle , Humains , Mâle , Souris , Amino-2 phosphono-5 valérate/effets indésirables , Amino-2 phosphono-5 valérate/métabolisme , Amino-2 phosphono-5 valérate/pharmacologie , Canaux ioniques sensibles à l'acidité/composition chimique , Canaux ioniques sensibles à l'acidité/déficit , Canaux ioniques sensibles à l'acidité/effets des médicaments et des substances chimiques , Canaux ioniques sensibles à l'acidité/génétique , Canaux ioniques sensibles à l'acidité/métabolisme , Régulation allostérique/effets des médicaments et des substances chimiques , Sites de fixation/génétique , Encéphalopathie ischémique/induit chimiquement , Encéphalopathie ischémique/traitement médicamenteux , Encéphalopathie ischémique/métabolisme , Encéphalopathie ischémique/anatomopathologie , Modèles animaux de maladie humaine , Évaluation préclinique de médicament , Acide glutamique/analogues et dérivés , Acide glutamique/métabolisme , Acide glutamique/pharmacologie , Acide glutamique/toxicité , Souris knockout , Mutagenèse dirigée , Protons , Récepteurs du N-méthyl-D-aspartate/antagonistes et inhibiteurs , Récepteurs du N-méthyl-D-aspartate/composition chimique , Récepteurs du N-méthyl-D-aspartate/métabolismeRÉSUMÉ
Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.
Sujet(s)
Héparine , Héparitine sulfate , Héparitine sulfate/composition chimique , Héparitine sulfate/immunologie , Héparitine sulfate/métabolisme , Héparine/composition chimique , Héparine/métabolisme , Simulation de docking moléculaire , Anticorps à chaîne unique/composition chimique , Anticorps à chaîne unique/immunologie , Anticorps à chaîne unique/génétique , Humains , Animaux , Mutagenèse dirigée , Sites de fixation , Séquence d'acides aminésRÉSUMÉ
Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon ß-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of ß-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.
Sujet(s)
Simulation de dynamique moléculaire , Liaison aux protéines , Récepteurs CXCR , Humains , Récepteurs CXCR/métabolisme , Récepteurs CXCR/génétique , Sites de fixation , Conformation des protéines , bêta-Arrestine 1/métabolisme , bêta-Arrestine 1/génétique , Ligands , Cellules HEK293 , Mutagenèse dirigée , Régulation allostérique , Relation structure-activitéRÉSUMÉ
BACKGROUND: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment. METHODS AND RESULTS: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20. CONCLUSION: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies.
Sujet(s)
Affinité des anticorps , Antigènes CD20 , Régions déterminant la complémentarité , Mutagenèse dirigée , Rituximab , Anticorps à domaine unique , Anticorps à domaine unique/génétique , Anticorps à domaine unique/immunologie , Mutagenèse dirigée/méthodes , Antigènes CD20/immunologie , Antigènes CD20/génétique , Antigènes CD20/métabolisme , Humains , Rituximab/pharmacologie , Régions déterminant la complémentarité/génétique , Régions déterminant la complémentarité/immunologie , Lignée cellulaire tumorale , AnimauxRÉSUMÉ
The intricate mechanisms in the biosynthesis of terpenes belong to the most challenging problems in natural product chemistry. Methods to address these problems include the structure-based site-directed mutagenesis of terpene synthases, computational approaches, and isotopic labeling experiments. The latter approach has a long tradition in biosynthesis studies and has recently experienced a revival, after genome sequencing enabled rapid access to biosynthetic genes and enzymes. Today, this allows for a combined approach in which isotopically labeled substrates can be incubated with recombinant terpene synthases. These clearly defined reaction setups can give detailed mechanistic insights into the reactions catalyzed by terpene synthases, and recent developments have substantially deepened our understanding of terpene biosynthesis. This chapter will discuss the state of the art and introduce some of the most important methods that make use of isotopic labelings in mechanistic studies on terpene synthases.
Sujet(s)
Alkyl et aryl transferases , Marquage isotopique , Terpènes , Alkyl et aryl transferases/métabolisme , Alkyl et aryl transferases/génétique , Alkyl et aryl transferases/composition chimique , Marquage isotopique/méthodes , Terpènes/métabolisme , Terpènes/composition chimique , Mutagenèse dirigée/méthodes , Protéines recombinantes/métabolisme , Protéines recombinantes/génétique , Protéines recombinantes/composition chimiqueRÉSUMÉ
α-Synuclein (α-syn) is a small protein that is involved in cell vesicle trafficking in neuronal synapses. A progressive aggregation of this protein is the expected molecular cause of Parkinson's disease, a disease that affects millions of people around the world. A growing body of evidence indicates that phospholipids can strongly accelerate α-syn aggregation and alter the toxicity of α-syn oligomers and fibrils formed in the presence of lipid vesicles. This effect is attributed to the presence of high copies of lysines in the N-terminus of the protein. In this study, we performed site-directed mutagenesis and replaced one out of two lysines at each of the five sites located in the α-syn N-terminus. Using several biophysical and cellular approaches, we investigated the extent to which six negatively charged fatty acids (FAs) could alter the aggregation properties of K10A, K23A, K32A, K43A, and K58A α-syn. We found that FAs uniquely modified the aggregation properties of K43A, K58A, and WT α-syn, as well as changed morphology of amyloid fibrils formed by these mutants. At the same time, FAs failed to cause substantial changes in the aggregation rates of K10A, K23A, and K32A α-syn, as well as alter the morphology and toxicity of the corresponding amyloid fibrils. Based on these results, we can conclude that K10, K23, and K32 amino acid residues play a critical role in protein-lipid interactions since their replacement on non-polar alanines strongly suppressed α-syn-lipid interactions.
Sujet(s)
Mutagenèse dirigée , alpha-Synucléine , alpha-Synucléine/métabolisme , alpha-Synucléine/génétique , Humains , Amyloïde/métabolisme , Amyloïde/génétique , Acides gras/métabolismeRÉSUMÉ
Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.