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Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated. Supplementary Information: The online version contains supplementary material available at 10.1007/s00723-023-01618-8.
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HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 Å resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. Double electron-electron resonance spectroscopy provided further evidence for enriched occupancy of more open Env conformations. Consistent with conformational flexibility, 1059 Envs were associated with resistance to most bnAbs that exhibit reduced potency against functional Env intermediates. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal, typically occluded, epitopes. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that poorly recognize functional Env intermediates. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.
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Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.
Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Animais , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Neutralizantes/ultraestrutura , Sítios de Ligação , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenho de Fármacos , Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/uso terapêutico , Anticorpos Anti-HIV/ultraestrutura , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Macaca , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.
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Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Conformação Proteica , Biotinilação , Membrana Celular , Microscopia Crioeletrônica , Proteínas de Ligação a DNA , Endopeptidases , Escherichia coli , Proteínas de Escherichia coli/química , Modelos Moleculares , Desnaturação Proteica , Dobramento de Proteína , EstreptavidinaRESUMO
AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the ß-subunit. Here, using double electron-electron resonance (DEER) spectroscopy, we demonstrate that the CBM-KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD-CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD-CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.
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Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Regulação Alostérica , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzoatos/química , Benzoatos/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligantes , Conformação Proteica , Domínios ProteicosRESUMO
HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.
Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos CD4/metabolismo , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular , Espectroscopia de Ressonância de Spin Eletrônica , Epitopos/imunologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/genética , HumanosRESUMO
Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu(2+) bound to an engineered Cu(2+) binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.
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Espectroscopia de Ressonância de Spin Eletrônica , Proteínas/química , Marcadores de Spin , Sítios de Ligação , TemperaturaRESUMO
The intrinsically disordered protein (IDP) stathmin plays an important regulatory role in cytoskeletal maintenance through its helical binding to tubulin and microtubules. However, it lacks a stable fold in the absence of its binding partner. Although stathmin has been a focus of research over the past two decades, the solution-phase conformational dynamics of this IDP are poorly understood. It has been reported that stathmin is purely monomeric in solution and that it bears a short helical region of persistent foldedness, which may act to nucleate helical folding in the C-terminal direction. Here we report a comprehensive study of the structural equilibria local to this region in stathmin that contradicts these two claims. Using the technique of electron paramagnetic resonance (EPR) spectroscopy on spin-labeled stathmin mutants in the solution-phase and when immobilized on Sepharose solid support, we show that all sites in the helical nucleation region of stathmin exhibit multiple spectral components that correspond to dynamic states of differing mobilities and stabilities. Importantly, a state with relatively low mobility dominates each spectrum with an average population greater than 50%, which we suggest corresponds to an oligomerized state of the protein. This is in contrast to a less populated, more mobile state, which likely represents a helically folded monomeric state of stathmin, and a highly mobile state, which we propose is the random coil conformer of the protein. Our interpretation of the EPR data is confirmed by further characterization of the protein using the techniques of native and SDS PAGE, gel filtration chromatography, and multiangle and dynamic light scattering, all of which show the presence of oligomeric stathmin in solution. Collectively, these data suggest that stathmin exists in a diverse equilibrium of states throughout the purported helical nucleation region and that this IDP exhibits a propensity toward oligomerization.
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Proteínas Intrinsicamente Desordenadas/química , Estatmina/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Estatmina/metabolismo , TermodinâmicaRESUMO
Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron-electron resonance (DEER), is carried out at cryogenic temperatures (50-80 K) to increase the short spin-spin relaxation time (T2) upon which the technique relies. A challenge is to measure long-range distances (20-60 Å) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin-lattice relaxation rate (T1(-1)) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu(2+), it has been found that interspin distances ≤25 Å can be determined in this way (Jun et al. Biochemistry 2006, 45, 11666). Here, the upper limit of the accessible distance is extended to ≈40 Å using spin labels with long T1, a high-affinity 5-residue Cu(2+) binding loop inserted into the protein sequence, and pulsed saturation recovery to measure relaxation enhancement. Time-domain Cu(2+) electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu(2+) loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu(2+)/nitroxide DEER at cryogenic temperatures and T1 relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.
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Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas/química , Temperatura , Sequência de Aminoácidos , Bacteriófago T4/enzimologia , Sítios de Ligação , Cobre/metabolismo , Modelos Moleculares , Muramidase/química , Muramidase/genética , Muramidase/metabolismo , Mutação , Óxidos de Nitrogênio/química , Peptídeos/química , Estrutura Secundária de Proteína , Proteínas/genética , Proteínas/metabolismo , Teoria Quântica , Rotação , Marcadores de SpinRESUMO
The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a K(m) of 3.35 ± 0.31 µM, a k(cat) of 0.0717 ± 0.0016 s(-1) and a k(cat)/K(m) of 21,400 ± 2000 M(-1) s(-1).
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Antivirais/química , Antivirais/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Vírus do Nilo Ocidental/enzimologia , Descoberta de Drogas , Ensaios Enzimáticos/métodos , Humanos , Modelos Moleculares , Febre do Nilo Ocidental/tratamento farmacológicoRESUMO
A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an α-helix, at i and i + 2 in a ß-strand, or with cysteine residues in adjacent strands in a ß-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.
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Óxidos de Nitrogênio/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Conformação ProteicaRESUMO
Reported here is the first µSR study of the muon (A(µ)) and proton (A(p)) ß-hyperfine coupling constants (Hfcc) of muoniated sec-butyl radicals, formed by muonium (Mu) addition to 1-butene and to cis- and trans-2-butene. The data are compared with in vacuo spin-unrestricted MP2 and hybrid DFT/B3YLP calculations reported in the previous paper (I), which played an important part in the interpretation of the data. The T-dependences of both the (reduced) muon, A(µ)'(T), and proton, A(p)(T), Hfcc are surprisingly well explained by a simple model, in which the calculated Hfcc from paper I at energy minima of 0 and near ±120° are thermally averaged, assuming an energy dependence given by a basic 2-fold torsional potential. Fitted torsional barriers to A(µ)'(T) from this model are similar (~3 kJ/mol) for all muoniated butyl radicals, suggesting that these are dominated by ZPE effects arising from the C−Mu bond, but for A(p)(T) exhibit wide variations depending on environment. For the cis- and trans-2-butyl radicals formed from 2-butene, A(µ)'(T) exhibits clear discontinuities at bulk butene melting points, evidence for molecular interactions enhancing these muon Hfcc in the environment of the solid state, similar to that found in earlier reports for muoniated tert-butyl. In contrast, for Mu−sec-butyl formed from 1-butene, there is no such discontinuity. The muon hfcc for the trans-2-butyl radical are seemingly very well predicted by B3LYP calculations in the solid phase, but for sec-butyl from 1-butene, showing the absence of further interactions, much better agreement is found with the MP2 calculations across the whole temperature range. Examples of large proton Hfcc near 0 K are also reported, due to eclipsed C−H bonds, in like manner to C−Mu, which then also exhibit clear discontinuities in A(p)(T) at bulk melting points. The data suggest that the good agreement found between theory and experiment from the B3LYP calculations for eclipsed bonds in the solid phase may be fortuitous. For the staggered protons of the sec-butyl radicals formed, no discontinuities are seen at all in A(p)(T), also demonstrating no further effects of molecular interactions on these particular proton Hfcc.
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The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ≈0.1-1 µs range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by saturation recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1-70 µs can be estimated by the approach.