Biochemistry Biochemistry最新文献

Exploring the nature of histidine residue tautomerization via a systematic conformational study is essential for understanding the pathology and toxicity of several neurodegenerative diseases, as well as for their diagnosis and treatment. Herein, density functional theory (DFT) calculations were used to determine the Tau protein's histidine-containing dipeptide (Lys-His, His-Gln, and His-Val) and tripeptide (Lys-His-Gln and Lys-His-Val) isomeric conformations via intramolecular hydrogen bond interactions, with particular attention to the influence of N-H group isomeric forms on their properties. The calculated infrared (IR) spectroscopy of the N-H stretch region of each isomer and nuclear magnetic resonance (NMR) shielding of the imidazole ring carbon atoms (¹³C¹, ¹³C², and ¹³C³) were investigated. The results show that both the IR spectrum of the N-H group and the NMR shielding of ¹³C nuclei on the imidazole ring can be used to identify the histidine-containing dipeptide and tripeptide tautomeric isomers. Systematically analyzing the hydrogen bonding interactions, the atomic charge distribution, the potential energy distribution, and the HOMO-LUMO transitions of each isomer further verified the above conclusions. This study provides theoretical evidence for the conformation identification of the histidine-containing dipeptide and tripeptide isomers on Tau protein.

Oomycete pathogens cause large economic losses in agriculture through diseases such as late blight (Phytophthora infestans), and stem and root rot of soybean (Phytophthora sojae). The effector protein AVR3a, from P. infestans, and its homologue AVR3a11 from Phytophthora capsici, are host-translocated effectors that interact with plant proteins to evade defense mechanisms and enable infection. Both proteins belong to the family of RXLR effectors and contain an N-terminal secretion signal, an RXLR motif for translocation into the host cell, and a C-terminal effector domain. Within this family, many proteins have been predicted to contain one or more WY domains as their effector domain, which is proposed to encompass a conserved minimal core fold containing three helices, further stabilized by additional helices or dimerization. In AVR3a11, a helical N-terminal extension to the core fold forms a four-helix bundle, as determined by X-ray crystallography. For a complete picture of the dynamics of AVR3a11, we have determined the solution structure of AVR3a11, and studied its dynamics in the fast time scale (ns-ps, from NMR relaxation parameters) and in the slow time scale (seconds to minutes, from hydrogen/deuterium exchange experiments). Hydrogen/deuterium exchange showed that the N-terminal helix is less stable than the other three helices, confirming the core fold originally proposed. Relaxation measurements confirm that AVR3a11 undergoes extensive conformational exchange, despite the uniform presence of fast motions in the spectral density function throughout most of its sequence. As functional residues are in the more mobile regions, flexibility in the slow/intermediate time scale may be functionally important.

Membrane binding of monotopic proteins can involve various post-translational modifications or a combination of some membrane-binding elements. For example, amphipathic α helices and palmitoylation could drive the membrane attachment of proteins. G-protein-coupled receptor kinases (GRKs) regulate the activity of G-protein-coupled receptors. Several members of the family of GRKs are acylated. Moreover, the C-terminus of GRK6 contains an amphipathic α helix and a palmitoyl group, which could also be the case for GRK4 isoforms. In our experiments, GRK4α/β-derived peptides of differing C-terminal lengths (Cter-GRK4α/β variants) were thus studied to discriminate the individual role of the palmitoyl group and amphipathic α helix of Cter-GRK4α/β in its membrane binding. The membrane binding of the Cter-GRK4α/β variants was studied by comparing their maximum insertion pressure (MIP) to lipid monolayers as well as their intrinsic fluorescence properties using large unilamellar vesicles. The MIP data show a higher level of binding of the palmitoylated longest GRK4α/β variant. Moreover, MIP measurements in the absence and presence of 15 mol % of the negatively charged phosphoserine demonstrated that the amphipathic α helix of Cter-GRK4α/β plays a major role in its membrane binding. Accordingly, partition studies of the Cter-GRK4α/β variants to membranes by fluorescence spectroscopy demonstrate the involvement of the palmitoyl group and the amphipathic α helix of the C-terminus of GRK4α/β in its membrane binding. Altogether, the data show that both the palmitoyl group and the amphipathic helix highly favor membrane binding of the C-terminus of GRK4α/β, which should facilitate the proper anchoring of GRK4α/β and phosphorylation of GPCRs.

We describe the capture and structuring of disordered N-terminal regions by the macrocycle sulfonato-calix[4]arene (sclx₄). Using the trimeric β-propeller Ralstonia solanacearum lectin (RSL) as a scaffold, we generated a series of mutants with extended and dynamic N-termini. Three of the mutants feature an N-terminal methionine-lysine motif. The fourth mutant contains the disordered 8-residue N-terminus of Histone 3, a component of the nucleosome. X-ray crystallography and NMR spectroscopy provide evidence for sclx₄ binding to the flexible N-terminal regions. Three crystal structures reveal that the calixarene recognizes the N-terminal Met-Lys motif, capturing either residue. We provide crystallographic proof for sclx₄ encapsulation of N-terminal methionine. Calixarene capture of intrinsically disordered regions may have applications in regulating protein secondary (and tertiary) structure.

Translation initiation represents a critical regulatory step in gene expression, orchestrated by the interaction of eukaryotic initiation factor 4E (eIF4E) with the 7-methylguanosine (m⁷G) cap structure at the 5' end of mRNA. This interaction enables eIF4F, composed of eIF4E, eIF4G, and eIF4A, to recruit the 43S preinitiation complex to the mRNA 5' end. The activity of eIF4E is tightly regulated and often dysregulated in cancer, neurological disorders, and viral infections. To investigate the interactions of human eIF4E with m⁷G-RNA and eIF4G, we engineered single-cysteine mutants of eIF4E to enable fluorescent dye attachment. However, these mutants presented challenges in purification and exhibited diminished activity. To overcome these issues, we developed a method to fluorescently label eIF4E via sortase-mediated transpeptidation. Our results demonstrate that sortase-labeled eIF4E retains activity comparable to wild-type eIF4E. This approach provides a valuable tool for studying the dynamic mechanisms of translation initiation and its regulation.

Listeriolysin O (LLO) is a potent membrane-damaging pore-forming toxin (PFT) secreted by the bacterial pathogen Listeria monocytogenes. LLO belongs to the family of cholesterol-dependent cytolysins (CDCs), which specifically target cholesterol-containing cell membranes to form oligomeric pores and induce membrane damage. CDCs, including LLO, harbor designated pore-forming motifs. In the soluble monomeric state, these motifs are present as helical segments (two transmembrane helices (TMHs); TMH1 and TMH2), and in the course of oligomeric pore formation, they convert into transmembrane β-hairpins to form the β-barrel scaffold of the CDC pores. Despite their well-established role in forming the β-barrel pore scaffold, precise structural implications of the two distinct TMH motifs and their membrane-insertion mechanism still remain obscure. Here, we show that the two TMH motifs of LLO contribute differently to maintaining the structural integrity of the toxin. While the deletion of TMH1 imposed a more serious defect, truncation of TMH2 was found to have a less severe effect on the structural integrity. Despite showing membrane-binding and oligomerization ability, the TMH2-deleted LLO variant displayed drastically abrogated pore-forming activity, presumably due to compromised membrane-insertion efficacy of the pore-forming TMH motifs. When probed for the membrane-insertion mechanism, we found slower membrane-insertion kinetics for TMH2 than for TMH1. Interestingly, deletion of TMH2 arrested membrane insertion of TMH1, thus suggesting a stringent cooperation between the two TMH motifs in regulating the pore-formation mechanism of LLO. Taken together, our study provides new mechanistic insights regarding the membrane-damaging action of LLO, in the CDC family of PFTs.

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.

The peridinin-chlorophyll a-protein is a light-harvesting complex found in dinoflagellates, which has an unusually high fraction of carotenoids. The carotenoids are directly involved in the energy transfer to chlorophyll with high efficiency. The detailed mechanism of energy transfer and the roles of the protein in the process remain debated in the literature, in part because most calculations have focused on a limited number of chromophore structures. Here we investigate the magnitude of the fluctuations of the site energies of individual and coupled chromophores, as the results are essential to the understanding of experimental spectra and the energy transfer mechanism. To this end, we sampled conformations of the PCP complex by means of classical and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations. Subsequently we performed (supermolecular) excitation energy calculations on a statistically significant number of snapshots using TD-LC-DFT/CAM-B3LYP and the semiempirical time-dependent long-range corrected density functional tight binding (TD-LC-DFTB2) as the QM method. We observed that the magnitude of the site energy fluctuations is large compared to the differences of the site energies between the chromophores, and this also holds for the coupled chromophores. We also investigated the composition of the coupled states, the effect of coupling on the absorption spectra, as well as transition dipole moment orientations and the possibility of delocalized states with Chl a. Our study thus complements previous computational studies relying on a single structure and establishes the most prominent features of the coupled chromophores that are essential to the robustness of the energy transfer process.

Adhesion G protein-coupled receptors (ADGRs) belong to Class B2 of GPCRs and are involved in a wide array of important physiological processes. ADGRs contain a GPCR autoproteolysis-inducing domain that is proximal to the receptor N-terminus and undergoes autoproteolysis during the biosynthesis to generate two fragments: the N-terminal fragment (NTF) and the C-terminal fragment (CTF). Dissociation of NTF reveals a tethered agonist to activate the CTF of ADGRs for G protein signaling. Synthetic peptides that mimic the tethered agonist can also activate ADGRs. However, mechanisms of peptide agonist dissociation and the deactivation of ADGRs remain poorly understood. In this study, we have performed all-atom enhanced sampling simulations using a novel protein-protein interaction Gaussian-accelerated molecular dynamics (PPI-GaMD) method on the ADGRG2-IP15 and ADGRG1-P7 complexes. The PPI-GaMD simulations captured the dissociation of the IP15 and P7 peptide agonists from their target receptors. We were able to identify important low-energy conformations of ADGRG2 and ADGRG1 in the active, intermediate, and inactive states, as well as different states of the peptide agonists IP15 and P7 during dissociation. Therefore, our PPI-GaMD simulations have revealed dynamic mechanisms of peptide agonist dissociation and deactivation of ADGRG1 and ADGRG2, which will facilitate the rational design of peptide regulators of the two receptors and other ADGRs.

Iron-sulfur clusters are essential protein cofactors synthesized in human mitochondria by an NFS1-ISD11-ACP-ISCU2-FXN assembly complex. Surprisingly, researchers have discovered three distinct quaternary structures for cysteine desulfurase subcomplexes, which display similar interactions between NFS1-ISD11-ACP protomeric units but dramatically different dimeric interfaces between the protomers. Although the role of these different architectures is unclear, possible functions include regulating activity and promoting the biosynthesis of distinct sulfur-containing biomolecules. Here, crystallography, native ion-mobility mass spectrometry, and chromatography methods reveal the Fe-S assembly subcomplex exists as an equilibrium mixture of these different quaternary structures. Isotope labeling and native mass spectrometry experiments show that the NFS1-ISD11-ACP complexes disassemble into protomers, which can then undergo exchange reactions and dimerize to reform native complexes. Single crystals isolated in distinct architectures have the same activity profile and activation by the Friedreich's ataxia (FRDA) protein frataxin (FXN) when rinsed and dissolved in assay buffer. These results suggest FXN functions as a "molecular lock" and shifts the equilibrium toward one of the architectures to stimulate the cysteine desulfurase activity and promote iron-sulfur cluster biosynthesis. An NFS1-designed variant similarly shifts the equilibrium and partially replaces FXN in activating the complex. We propose that eukaryotic cysteine desulfurases are unusual members of the morpheein class of enzymes that control their activity through their oligomeric state. Overall, the findings support architectural switching as a regulatory mechanism linked to FXN activation of the human Fe-S cluster biosynthetic complex and provide new opportunities for therapeutic interventions of the fatal neurodegenerative disease FRDA.