Biochemistry Biochemistry最新文献

The receptor tyrosine kinase EphB4 is involved in tumor angiogenesis, proliferation, and metastasis. Designed ankyrin repeat proteins (DARPins) binding to the EphB4 extracellular domain were identified from a combinatorial library using phage display. Surface plasmon resonance (SPR) allowed us to distinguish between DARPins that either compete with the EphB4 ligand ephrin-B2 for binding to a common site or target a different epitope. The identified DARPins all prevent ligand-induced EphB4 phosphorylation and impair tube formation by endothelial cells in vitro. The competitive DARPin AB1 was additionally shown to inhibit vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-induced angiogenesis in vivo. In summary, we have isolated DARPins that exert antiangiogenic effects by specifically binding to EphB4 and may potentially lead to new cancer therapeutics.

Eukaryotic Initiation Factor 4 (eIF4) is a group of factors that activates mRNA for translation and recruit 43S preinitiation complex (PIC) to the mRNA 5' end, forming the 48S PIC. The eIF4 factors include mRNA 5' cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffold protein eIF4G, which anchors eIF4A and eIF4E. Another eIF4 factor, eIF4B, stimulates the RNA helicase activity of eIF4A and facilitates mRNA recruitment. However, the mechanisms by which eIF4B binds the 40S ribosomal subunit and promotes mRNA recruitment remain poorly understood. Using cryo-Eletron Microscopy (cryo-EM), we obtained a map of the yeast 40S ribosomal subunit in a complex with eIF4B (40S-eIF4B complex). An extra density, tentatively assigned to yeast eIF4B, was observed near the mRNA entry channel of the 40S, contacting ribosomal proteins uS10, uS3, and eS10 as well as rRNA helix h16. Predictive modeling of the 40S-eIF4B complex suggests that the N-terminal domain of eIF4B binds near the mRNA entry channel, overlapping with the extra density observed in the 40S-eIF4B map. The partially open conformation of 40S in the 40S-eIF4B map is incompatible with eIF3j binding observed in the 48S PIC. Additionally, the extra density at the mRNA entry channel poses steric hindrance for eIF3g binding in the 48S PIC. Thus, structural insights suggest that eIF4B facilitates the release of eIF3j and the relocation of the eIF3b-g-i module during mRNA recruitment, thereby advancing our understanding of eIF4B's role in translation initiation.

Human CblC catalyzes the indispensable processing of dietary vitamin B₁₂ by the removal of its β-axial ligand and an either one- or two-electron reduction of its cobalt center to yield cob(II)alamin and cob(I)alamin, respectively. Human CblC possesses five cysteine residues of an unknown function. We hypothesized that Cys149, conserved in mammals, tunes the CblC reactivity. To test this, we recreated an evolutionary early variant of CblC, namely, Cys149Ser, as well as Cys149Ala. Surprisingly, substitution of Cys149 for serine or alanine led to faster observed rates of glutathione-driven dealkylation of MeCbl compared to wild-type CblC. The reaction yielded aquacobalamin and stoichiometric formation of S-methylglutathione as the demethylation products. Determination of end-point oxidized glutathione revealed significantly uncoupled electron transfer in both mutants compared with the wild type. Long incubation times revealed the conversion of aquacobalamin to cob(II)alamin in the presence of oxygen in mutants Cys149Ser and Cys149Ala but not in wild-type CblC, all without an effect on dealkylation rates. This finding is reminiscent of the catalytic behavior of CblC from Caenorhabditis elegans, wherein Cys149 is naturally substituted by Ser, and the reaction mechanism differs from that of human CblC precisely by the unusual stabilization of cob(II)alamin in the presence of oxygen. Thus, Cys149 tunes the catalytic activity of human CblC by minimizing uncoupled electron transfer that forms GSSG. This occurs at the expense of a slower observed rate constant for the demethylation of MeCbl. This adjustment is compatible with diminished needs for intracellular turnover of cobalamins and with life under increased oxygen concentration.

Endonuclease III (EndoIII), a key enzyme in the base excision repair (BER) pathway, contains a [4Fe4S] cluster that facilitates DNA repair through DNA-mediated charge transfer. Recent findings indicate that the redox state of this cluster influences EndoIII's binding affinity for DNA, modulating the enzyme's activity. In this study, we investigated the structural and electronic changes of the [4Fe4S] cluster upon binding to double-stranded DNA (dsDNA) using Fourier transform infrared spectroscopy, density functional theory calculations, and machine learning models. Our results reveal shifts in Fe-S bond vibrational modes, suggesting stabilization of the oxidized [4Fe4S] cluster in proximity to negatively charged DNA. A machine learning model, trained on the spectral features of the EndoIII/DNA complex, predicted the enzyme-DNA binding distance, providing further insights into the structural changes upon binding. We correlated the electrochemical stabilization potential of 150 mV in the [4Fe4S] cluster with the enzyme's DNA-binding properties, demonstrating how the cluster's redox state plays a crucial role in both structural stability and DNA repair.

G-protein-coupled receptors (GPCRs) transmit an extracellular chemical/biological signal across the cell membrane, stimulating an array of intracellular signaling cascades. Canonically, these extracellular signaling molecules bind to the endogenous ligand pocket (orthosteric pocket), which stabilizes either an active or inactive conformational ensemble of the receptor. However, recent structural evidence indicates that small molecules can mediate the protein-protein interactions between the GPCR and their intracellular transducers. These small molecules are reminiscent of molecular glues and can be powerful tools for modulating GPCR signaling bias. In this Perspective, we will investigate the current structural information available on molecular glues and how they modulate GPCR signaling bias. We also examine the prospects of molecular glues and GPCR drug/probe design.

Understanding the mechanisms of allosteric regulation in response to second messengers is crucial for advancing basic and applied research. This study focuses on the differential allosteric regulation by the ubiquitous signaling molecule, cAMP, in the cAMP receptor protein from Escherichia coli (CRP_Ecoli) and from Mycobacterium tuberculosis (CRP_MTB). By introducing structurally homologous mutations from allosteric hotspots previously identified in CRP_Ecoli into CRP_MTB and examining their effects on protein solution structure, stability and function, we aimed to determine the factors contributing to their differential allosteric regulation. Our results demonstrate that the mutations did not significantly alter the overall fold, assembly and thermodynamic stability of CRP_MTB, but had varying effects on cAMP binding affinity and cooperativity. Interestingly, the mutations had minimal impact on the specific binding of CRP_MTB to DNA promoter sites. However, we found that cAMP primarily reduces nonspecific CRP_MTB-DNA complexes and that the mutants largely lose this ability. Furthermore, our experiments revealed that CRP_MTB-DNA complexes serve as a nucleation point for additional binding of CRP_MTB proteins to form high-order oligomers with the DNA. Overall, our findings highlight the importance of both cAMP and DNA interactions in modulating the allosteric regulation of CRP_MTB and provide insights into the differential responses of CRP_Ecoli and CRP_MTB to cAMP.

SARS-CoV-2 variant recurrence has emphasized the imperative prerequisite for effective antivirals. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication, making it one of the prime and promising antiviral targets. Mpro features several druggable sites, including active sites and allosteric sites near the dimerization interface, that regulate its catalytic activity. This study identified six highly efficacious antiviral SARS-CoV-2 compounds (WIN-62577, KT185, bexarotene, ledipasvir, diacerein, and simepervir) using structure-based virtual screening of compound libraries against Mpro. Using SPR and ITC, the binding of selected inhibitory compounds to the target Mpro was validated. The FRET-based protease assay demonstrated that the identified molecules effectively inhibit Mpro with IC₅₀ values in the range from 0.64 to 11.98 μM. Additionally, in vitro cell-based antiviral assays showed high efficacy with EC₅₀ values in the range of 1.51 to 18.92 μM. The crystal structure of the Mpro-minocycline complex detailed the possible inhibition mechanism of minocycline, an FDA-approved antibiotic. Minocycline binds to an allosteric site, revealing residues critical for the loss of protease activity due to destabilization of molecular interactions at the dimeric interface, which are crucial for the proteolytic activity of Mpro. The study suggests that the binding of minocycline to the allosteric site may play a role in Mpro dimer destabilization and direct the rational design of minocycline derivatives as antiviral drugs.

SoxR containing a [2Fe-2S] cluster required for its transcription activity functions as a bacterial stress-response sensor that is activated through oxidation by redox-active compounds (RACs). SoxR from Escherichia coli (EcSoxR) is activated by nearly all RACs nonspecifically. In contrast, nonenteric SoxRs such as Pseudomonas aeruginosa (PaSoxR), and Streptomyces coelicolor (ScSoxR) activate their target genes in response to RAC including endogenously produced metabolites. To investigate the determinants of SoxR's activity, the endogenous or various synthetic RACs-mediated oxidation of the [2Fe-2S] cluster of EcSoxR, PaSoxR, and ScSoxR were measured by pulse radiolysis. Radiolytically generated hydrated electrons (e_aq^-) very rapidly reduced the oxidized form of the [2Fe-2S] cluster of SoxR. In the presence of RAC, a subsequent increase in absorption in the visible region corresponding to reoxidation of the [2Fe-2S] cluster was observed on a time scale of milliseconds. Both EcSoxR and PaSoxR reacted very rapidly (2.0 × 10⁸ to 2.0 × 10⁹ M^-1 s^-1) with various RACs, including viologen, phenazines, and quinones. No differences in kinetic behaviors were evident between EcSoxR and PaSoxR, whereas ScSoxR reacted with a limited range of RACs.

The enzyme 4-oxo-l-proline reductase (BDH2) has recently been identified in humans. BDH2, previously thought to be a cytosolic (R)-3-hydroxybutyrate dehydrogenase, actually catalyzes the NADH-dependent reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline, a compound with known anticancer activity. Here we provide an initial mechanistic characterization of the BDH2-catalyzed reaction. Haldane relationships show the reaction equilibrium strongly favors the formation of cis-4-hydroxy-l-proline. Stereospecific deuteration of NADH C4 coupled with mass spectrometry analysis of the reaction established that the pro-S hydrogen is transferred. NADH is co-purified with the enzyme, and a binding kinetics competition assays with NAD⁺ defined dissociation rate constants for NADH of 0.13 s^-1 at 5 °C and 7.2 s^-1 at 25 °C. Isothermal titration calorimetry at 25 °C defined equilibrium dissociation constants of 0.48 and 29 μM for the BDH2:NADH and BDH2:NAD⁺ complexes, respectively. Differential scanning fluorimetry showed BDH2 is highly thermostabilized by NADH and NAD⁺. The k_cat/K_M pH-rate profile indicates that a group with a pK_a of 7.3 and possibly another with a pK_a of 8.7 must be deprotonated and protonated, respectively, for maximum binding of 4-oxo-l-proline and/or catalysis, while the k_cat profile is largely insensitive to pH in the pH range used. The single-turnover rate constant is only 2-fold higher than k_cat. This agrees with a pre-steady-state burst of substrate consumption, suggesting that a step after chemistry, possibly product release, contributes to limit k_cat. A modest solvent viscosity effect on k_cat indicates that this step is only partially diffusional. Taken together, these data suggest chemistry does not limit the reaction rate but may contribute to it.

C-terminal amidation of antimicrobial peptides (AMPs) is a frequent minor modification used to improve antibacterial potency, commonly ascribed to increased positive charge, protection from proteases, and a stabilized secondary structure. Although the activity of AMPs is primarily associated with the ability to penetrate bacterial membranes, hitherto the effect of amidation on this interaction has not been understood in detail. Here, we show that amidation of the scorpion-derived membranolytic peptide AamAP1-Lys produces a potent analog with faster bactericidal activity, increased membrane permeabilization, and greater Gram-negative membrane penetration associated with greater conformational flexibility. AamAP1-lys-NH₂ has improved antibiofilm activity against Acinetobacter baumannii and Escherichia coli, benefits from a two- to 3-fold selectivity improvement, and provides protection against A. baumannii infection in a Galleria mellonella burn wound model. Circular dichroism spectroscopy shows both peptides adopt α-helix conformations in the steady state. However, molecular dynamics (MD) simulations reveal that, during initial binding, AamAP1-Lys-NH₂ has greater conformation heterogeneity, with substantial polyproline-II conformation detected alongside α-helix, and penetrates the bilayer more readily than AamAP1-Lys. AamAP1-Lys-NH₂ induced membrane permeabilization of A. baumannii occurs only above a critical concentration with slow and weak permeabilization and slow killing occurring at its lower MIC but causes greater and faster permeabilization than AamAP1-Lys, and kills more rapidly, when applied at equal concentrations. Therefore, while the increased potency of AamAP1-Lys-NH₂ is associated with slow bactericidal killing, amidation, and the conformational flexibility it induces, affords an improvement in the AMP pharmacodynamic profile and may need to be considered to achieve improved therapeutic performance.