RÉSUMÉ
The assembly of two monomeric constructs spanning segments 1-199 (MPro1-199) and 10-306 (MPro10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by â¼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro1-199 and MPro10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201-214 onto a second helical domain of MPro10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at â¼6 µM MPro1-199. Catalytic activity arises primarily from MPro1-199 and is dependent on the interface interactions involving the N-finger residues 1-9 of MPro1-199 and E290 of MPro10-306. Importantly, our results confirm that a single N-finger region with its associated inter-subunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.
RÉSUMÉ
The two most abundant isoforms of amyloid-ß (Aß) are the 40- (Aß40) and 42-residue (Aß42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aß aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aß42 and Aß40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aß42 with a small amount of Aß40, while the second phase results mostly from aggregation of Aß40. We also found that the aggregation of Aß42 is slowed by Aß40 while the aggregation of Aß40 is accelerated by Aß42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aß42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aß40 in the co-oligomers in a 1:1 mixture of Aß42 and Aß40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aß42 and Aß40 (1:10), suggesting the critical role of Aß40 in oligomer formation and aggregation.
RÉSUMÉ
N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 310-helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.
Sujet(s)
Domaine catalytique , Protéases 3C des coronavirus , Inhibiteurs des protéases des coronavirus , Indazoles , Multimérisation de protéines , SARS-CoV-2 , Triazines , Triazoles , Humains , Protéases 3C des coronavirus/métabolisme , Protéases 3C des coronavirus/composition chimique , Indazoles/composition chimique , Indazoles/pharmacologie , Modèles moléculaires , Mutation , Liaison aux protéines , Conformation des protéines , SARS-CoV-2/enzymologie , SARS-CoV-2/métabolisme , Triazines/composition chimique , Triazines/pharmacologie , Triazoles/composition chimique , Triazoles/pharmacologie , Inhibiteurs des protéases des coronavirus/composition chimique , Inhibiteurs des protéases des coronavirus/pharmacologieRÉSUMÉ
A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MProWT) from precursor polyproteins. Upon expression, a model precursor of MProWT mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MProM) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MProWT. MProM and the catalytic domain (MPro1-199) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MProM and MPro1-199, respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.
Sujet(s)
COVID-19 , SARS-CoV-2 , Humains , SARS-CoV-2/génétique , COVID-19/génétique , Mutation , Protéases 3C des coronavirus/génétiqueRÉSUMÉ
The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MProWT) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373), and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is â¼400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decrease in free energy (ΔG) and thermal stability (Tm), respectively, relative to MProWT. The H41A mutation results in a 20-fold increase in the dissociation constant (Kd), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and Tm, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MProH41A, whereas no significant change is observed in binding to MProWT. Structures of the four inhibitor complexes with MPro1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MProWT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MProWT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MProC145A also results in a significant, but smaller, increase in Kd and decrease in ΔG and Tm, relative to NMV.
Sujet(s)
COVID-19 , Inhibiteurs des protéases des coronavirus , SARS-CoV-2 , Humains , Carbone , Inhibiteurs des protéases des coronavirus/composition chimique , Inhibiteurs des protéases des coronavirus/pharmacologie , Lactames , Leucine , Simulation de docking moléculaire , Simulation de dynamique moléculaire , Nitriles , SARS-CoV-2/effets des médicaments et des substances chimiques , SARS-CoV-2/enzymologieRÉSUMÉ
Fibrils formed by the 42-residue amyloid-ß peptide (Aß42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures. Previous studies of Aß42 fibrils, including fibrils prepared entirely in vitro or extracted from brain tissue and using solid-state NMR (ssNMR) or cryogenic electron microscopy (cryo-EM) methods, have found polymorphs with differences in amino acid sidechain orientations, lengths of structurally ordered segments, and contacts between cross-ß subunit pairs within a single filament. Despite these differences, Aß42 molecules adopt a common S-shaped conformation in all previously described high-resolution Aß42 fibril structures. Here we report two cryo-EM-based structures of Aß42 fibrils that are qualitatively different, in samples derived from AD brain tissue by seeded growth. In type A fibrils, residues 12 to 42 adopt a ν-shaped conformation, with both intra-subunit and intersubunit hydrophobic contacts to form a compact core. In type B fibrils, residues 2 to 42 adopt an υ-shaped conformation, with only intersubunit contacts and internal pores. Type A and type B fibrils have opposite helical handedness. Cryo-EM density maps and molecular dynamics simulations indicate intersubunit K16-A42 salt bridges in type B fibrils and partially occupied K28-A42 salt bridges in type A fibrils. The coexistence of two predominant polymorphs, with differences in N-terminal dynamics, is supported by ssNMR data, as is faithful propagation of structures from first-generation to second-generation brain-seeded Aß42 fibril samples. These results demonstrate that Aß42 fibrils can exhibit a greater range of structural variations than seen in previous studies.
Sujet(s)
Maladie d'Alzheimer , Peptides bêta-amyloïdes , Humains , Peptides bêta-amyloïdes/métabolisme , Maladie d'Alzheimer/métabolisme , Spectroscopie par résonance magnétique , Encéphale/métabolisme , Conformation moléculaire , Amyloïde/composition chimique , Fragments peptidiques/métabolismeRÉSUMÉ
We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (Tm) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [(-6)MProH41A and (-6*)MProH41A, respectively], and its corresponding mature MProH41A were systematically examined. While the H41A mutation exerts negligible effect on Tm and dimer dissociation constant (Kdimer) of MProH41A, relative to the wild type MPro, both miniprecursors show a 4-5 °C decrease in Tm and > 85-fold increase in Kdimer as compared to MProH41A. The Kd for the binding of the covalent inhibitor GC373 to (-6*)MProH41A increases â¼12-fold, relative to MProH41A, concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers' oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1-2 and 302-306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.
Sujet(s)
Protéases 3C des coronavirus , Inhibiteurs des protéases des coronavirus , SARS-CoV-2 , Humains , Domaine catalytique , Cristallographie aux rayons X , SARS-CoV-2/effets des médicaments et des substances chimiques , SARS-CoV-2/enzymologie , Protéases 3C des coronavirus/composition chimique , Protéases 3C des coronavirus/génétique , Stabilité protéique , Mutation , Inhibiteurs des protéases des coronavirus/composition chimiqueRÉSUMÉ
The monomeric catalytic domain (residues 1-199) of SARS-CoV-2 main protease (MPro1-199) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro1-199 junction. We report the catalytic activity and the dissociation constants of MPro1-199 and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MProWT and MPro1-199 and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.
Sujet(s)
COVID-19 , Acides aminés , Domaine catalytique , Protéases 3C des coronavirus , Humains , Peptide hydrolases , Polyprotéines , SARS-CoV-2/génétiqueRÉSUMÉ
The global motions of ubiquitin, a model protein, on the surface of anisotropically tumbling 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG):1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles are described. The shapes of POPG:DHPC bicelles prepared with high molar ratios q of POPG to DHPC can be approximated by prolate ellipsoids, with the ratio of ellipsoid dimensions and dimensions themselves increasing with higher values of q. Adaptation of the nuclear magnetic resonance (NMR) relaxation-based approach that we previously developed for interactions of ubiquitin with spherical POPG liposomes (Ceccon, A. J. Am. Chem. Soc. 2016, 138, 5789-5792) allowed us to quantitatively analyze the variation in lifetime line broadening of NMR signals (ΔR2) measured for ubiquitin in the presence of q = 2 POPG:DHPC bicelles and the associated transverse spin relaxation rates (R2,B) of bicelle-bound ubiquitin. Ubiquitin, transiently bound to POPG:DHPC bicelles, undergoes internal rotation about an axis orthogonal to the surface of the bicelle and perpendicular to the principal axis of its rotational diffusion tensor on the low microsecond time scale (â¼3 µs), while the rotation axis itself wobbles in a cone on a submicrosecond time scale (≤ 500 ns).
Sujet(s)
Liposomes , Nanoparticules , Double couche lipidique/composition chimique , Spectroscopie par résonance magnétique/méthodes , UbiquitinesRÉSUMÉ
The envelope glycoprotein gp41 of the HIV-1 virus mediates its entry into the host cell. During this process, gp41 undergoes large conformational changes and the energy released in the remodeling events is utilized to overcome the barrier associated with fusing the viral and host membranes. Although the structural intermediates of this fusion process are attractive targets for drug development, no detailed high-resolution structural information or quantitative thermodynamic characterization are available. By measuring the dynamic equilibrium between the lipid-bound intermediate and the post-fusion six-helical bundle (6HB) states of the gp41 ectodomain in the presence of bilayer membrane mimetics, we derived both the reaction kinetics and energies associated with these two states by solution NMR spectroscopy. At equilibrium, an exchange time constant of about 12 seconds at 38 °C is observed, and the post-fusion conformation is energetically more stable than the lipid-bound state by 3.4 kcal mol-1. The temperature dependence of the kinetics indicates that the folding occurs through a high-energy transition state which may resemble a 5HB structure. The energetics and kinetics of gp41 folding in the context of membrane bilayers provide a molecular basis for an improved understanding of viral membrane fusion.
Sujet(s)
Protéine d'enveloppe gp41 du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Pénétration virale , Protéine d'enveloppe gp41 du VIH/composition chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Humains , Double couche lipidique , Résonance magnétique nucléaire biomoléculaire , Domaines protéiques , Pliage des protéines , Thermodynamique , Facteurs tempsRÉSUMÉ
Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease (Mpro) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the Mpro/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor's keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the Mpro active site adapt to the inhibitor, which appears to be an intrinsic property of Mpro. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with Mpro which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.
Sujet(s)
Traitements médicamenteux de la COVID-19 , SARS-CoV-2 , Antiviraux/composition chimique , Antiviraux/pharmacologie , Vaccins contre la COVID-19 , Protéases 3C des coronavirus , Cyclopropanes , Humains , Lactames , Leucine/analogues et dérivés , Nitriles , Proline/analogues et dérivés , Inhibiteurs de protéases/composition chimique , Inhibiteurs de protéases/pharmacologie , Inhibiteurs de protéases/usage thérapeutique , Sulfones , UréeRÉSUMÉ
The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.
Sujet(s)
Protéases 3C des coronavirus/métabolisme , Catalyse , Domaine catalytique , Dichroïsme circulaire , Protéases 3C des coronavirus/antagonistes et inhibiteurs , Protéases 3C des coronavirus/composition chimique , Protéases 3C des coronavirus/génétique , Modèles moléculaires , Mutation , Pyrrolidines/composition chimique , Acides sulfoniques/composition chimique , ThermodynamiqueRÉSUMÉ
SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145-His41 dyad, can help in guiding drug design. Here, a 2.0â Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3' by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.
RÉSUMÉ
Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure-activity relationship (SAR) study of noncovalent compounds that bind in the enzyme's substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor's P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.
Sujet(s)
Protéases 3C des coronavirus , Inhibiteurs de protéases , Acide orotique/analogues et dérivés , Pipérazines , Conformation des protéines , Électricité statiqueRÉSUMÉ
Entry of SARS-CoV-2 into a host cell is mediated by spike, a class I viral fusion protein responsible for merging the viral and host cell membranes. Recent studies have revealed atomic-resolution models for both the postfusion 6-helix bundle (6HB) and the prefusion state of spike. However, a mechanistic understanding of the molecular basis for the intervening structural transition, important for the design of fusion inhibitors, has remained elusive. Using nuclear magnetic resonance spectroscopy and other biophysical methods, we demonstrate the presence of α-helical, membrane-bound, intermediate states of spike's heptad repeat (HR1 and HR2) domains that are embedded at the lipid-water interface while in a slow dynamic equilibrium with the postfusion 6HB state. These results support a model where the HR domains lower the large energy barrier associated with membrane fusion by destabilizing the host and viral membranes, while 6HB formation actively drives their fusion by forcing physical proximity.
RÉSUMÉ
Previous studies have shown that racemic mixtures of 40- and 42-residue amyloid-ß peptides (d,l-Aß40 and d,l-Aß42) form amyloid fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (l-Aß40 and l-Aß42), suggesting a "chiral inactivation" approach to abrogating the neurotoxicity of Aß oligomers (Aß-CI). Here we report a structural study of d,l-Aß40 fibrils, using electron microscopy, solid-state nuclear magnetic resonance (NMR), and density functional theory (DFT) calculations. Two- and three-dimensional solid-state NMR spectra indicate molecular conformations in d,l-Aß40 fibrils that resemble those in known l-Aß40 fibril structures. However, quantitative measurements of 13C-13C and 15N-13C distances in selectively labeled d,l-Aß40 fibril samples indicate a qualitatively different supramolecular structure. While cross-ß structures in mature l-Aß40 fibrils are comprised of in-register, parallel ß-sheets, our data indicate antiparallel ß-sheets in d,l-Aß40 fibrils, with alternation of d and l molecules along the fibril growth direction, i.e., antiparallel "rippled sheet" structures. The solid-state NMR data suggest the coexistence of d,l-Aß40 fibril polymorphs with three different registries of intermolecular hydrogen bonds within the antiparallel rippled sheets. DFT calculations support an energetic preference for antiparallel alignments of the ß-strand segments identified by solid-state NMR. These results provide insight into the structural basis for Aß-CI and establish the importance of rippled sheets in self-assembly of full-length, naturally occurring amyloidogenic peptides.
Sujet(s)
Peptides bêta-amyloïdes/composition chimique , Amyloïde/composition chimique , Théorie de la fonctionnelle de la densité , Résonance magnétique nucléaire biomoléculaire , Humains , Microscopie électroniqueRÉSUMÉ
An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.
Sujet(s)
Drépanocytose , Préparations pharmaceutiques , Régulation allostérique , Drépanocytose/traitement médicamenteux , Hémoglobines/métabolisme , Humains , Cinétique , Oxygène , Liaison aux protéinesRÉSUMÉ
HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C-terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient-derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V-containing proteins. We identified multiple IxI/V-bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V-binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein-protein interactions.
Sujet(s)
Maladie de Charcot-Marie-Tooth/génétique , Protéines du choc thermique/composition chimique , Chaperons moléculaires/composition chimique , Adulte , Sites de fixation , Cellules cultivées , Cellules HeLa , Protéines du choc thermique/génétique , Protéines du choc thermique/métabolisme , Humains , Cellules souches pluripotentes induites/cytologie , Cellules souches pluripotentes induites/métabolisme , Mâle , Chaperons moléculaires/génétique , Chaperons moléculaires/métabolisme , Simulation de dynamique moléculaire , Motoneurones/cytologie , Motoneurones/métabolisme , Mutation faux-sens , Liaison aux protéines , Multimérisation de protéinesRÉSUMÉ
The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660-674 ) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2 O than H2 O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13 Cα chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.
Sujet(s)
Protéine d'enveloppe gp41 du VIH/composition chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/composition chimique , Peptides/composition chimique , Structure en hélice alpha/physiologie , HumainsRÉSUMÉ
We describe theory, experiments, and analyses of three-color Förster resonance energy transfer (FRET) spectroscopy for probing sub-millisecond conformational dynamics of protein folding and binding of disordered proteins. We devise a scheme that uses single continuous-wave laser excitation of the donor instead of alternating excitation of the donor and one of the acceptors. This scheme alleviates photophysical problems of acceptors such as rapid photobleaching, which is crucial for high time resolution experiments with elevated illumination intensity. Our method exploits the molecular species with one of the acceptors absent or photobleached, from which two-color FRET data is collected in the same experiment. We show that three FRET efficiencies and kinetic parameters can be determined without alternating excitation from a global maximum likelihood analysis of two-color and three-color photon trajectories. We implement co-parallelization of CPU-GPU processing, which leads to a significant reduction of the likelihood calculation time for efficient parameter determination.