Biochemistry Biochemistry最新文献

Climate and environmental changes have modified the habitats of fungal pathogens, inflicting devastating effects on livestock and crop production. Additionally, drug-resistant fungi are increasing worldwide, driving the urgent need to identify new molecular scaffolds for the development of antifungal agents for humans, animals, and plants. Poacic acid (PA), a plant-derived stilbenoid, was recently discovered to be a novel molecular scaffold that inhibits the growth of several fungi. Its antifungal activity has been associated with perturbation of the production/assembly of the fungal cell wall β-1,3-glucan, but its mode of action is not resolved. In this study, we investigated the antifungal activity of PA and its derivatives on a panel of yeast. PA had a fungistatic effect on S. cerevisiae and a fungicidal effect on plasma membrane-damaged Candida albicans mutants. Live cell fluorescence microscopy experiments revealed that PA increases chitin production and modifies its cell wall distribution. Chitin production and cell growth returned to normal after prolonged incubation. The antifungal activity of PA was reduced in the presence of exogenous chitin, suggesting that the potentiation of chitin production is a stress response that helps the yeast cell overcome the effect of this antifungal stilbenoid. Growth inhibition was also reduced by metal ions, indicating that PA affects the metal homeostasis. These findings suggest that PA has a complex antifungal mechanism of action that involves perturbation of the cell wall β-1,3-glucan production/assembly, chitin production, and metal homeostasis.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]₂₅ sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]₂₅ sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]₂₅ sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10–25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded “opened” and liganded “closed” forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo “opened” and an artificial generated apo “closed” structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-myo-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.

Ras-related nuclear protein (Ran) is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and a regulator of multiple cellular processes. In healthy cells, the GTP-bound form of Ran is concentrated at chromatin, creating a Ran•GTP gradient that provides the driving force for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. The Ran•GTP gradient is maintained by the regulator of chromatin condensation 1 (RCC1), a guanine nucleotide exchange factor that accelerates GDP/GTP exchange in Ran. RCC1 interacts with nucleosomes, which are the fundamental repeating units of eukaryotic chromatin. Here, we present a cryo-EM analysis of a trimeric complex composed of the nucleosome core particle (NCP), RCC1, and Ran. While the contacts between RCC1 and Ran in the complex are preserved compared with a previously determined structure of RCC1-Ran, our study reveals that RCC1 and Ran interact dynamically with the NCP and undergo rocking motions on the nucleosome surface. Furthermore, the switch 1 region of Ran, which plays an important role in mediating conformational changes associated with the substitution of GDP and GTP nucleotides in Ras family members, appears to undergo disorder–order transitions and forms transient contacts with the C-terminal helix of histone H2B. Nucleotide exchange assays performed in the presence and absence of NCPs are not consistent with an active role for nucleosomes in nucleotide exchange, at least in vitro. Instead, the nucleosome stabilizes RCC1 and serves as a hub that concentrates RCC1 and Ran to promote efficient Ran•GDP to Ran•GTP conversion.

AQP4-IgG is an autoantibody associated with neuromyelitis optica spectroscopic disorder (NMOSD), a central nervous system inflammatory disease that requires early diagnosis and treatment. We designed two fusion proteins, AQP4-DARPin1 and AQP4-DARPin2, comprising the complete antigenic epitopes of aquaporin-4 (AQP4) and the constant region of the scaffold protein DARPin. These fusion proteins were expressed and purified from Escherichia coli and coated on microplates to develop an efficient method for detecting AQP4-IgG. Molecular dynamics simulation revealed that the fusion of AQP4 extracellular epitopes with DARPin did not alter the main structure of DARPin. The purified AQP4-DARPins bound recombinant antibody rAb-53 (AQP4-IgG) with affinities of 135 and 285 nM, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation demonstrated that AQP4-DARPin1 specifically recognized AQP4-IgG in the NMOSD patient serum. AQP4-DARPin1 as a coated antigen showed higher ELISA signal and end point dilution ratio than full-length AQP4. Our AQP4-DARPin1-coated AQP4-IgG ELISA had 100% specificity and 90% sensitivity. These results indicate that AQP4-DARPin1, compared to existing detection strategies that use full-length or extracellular loop peptides of AQP4, provides a new and more effective approach to the ELISA detection of NMOSD.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by a macrolactam linkage between the N-terminus and the side chain of an internal aspartic acid or glutamic acid residue. Instead of adopting a branched-cyclic conformation, lasso peptides are “threaded”, with the C-terminal tail passing through the macrocycle to present a kinetically trapped rotaxane conformation. The availability of enhanced bioinformatics methods has led to a significant increase in the number of secondary modifications found on lasso peptides. To uncover new ancillary modifications in a targeted manner, a bioinformatic strategy was developed to discover lasso peptides with modifications to tryptophan. This effort identified numerous putative lasso peptide biosynthetic gene clusters with core regions of the precursor peptides enriched in tryptophan. Parsing of these tryptophan (Trp)-rich biosynthetic gene clusters uncovered several putative ancillary modifying enzymes, including halogenases and dimethylallyltransferases expected to act upon Trp. Characterization of two gene products yielded a lasso peptide with two 5-Cl-Trp modifications (chlorolassin) and another bearing 5-dimethylallyl-Trp and 2,3-didehydro-Tyr modifications (wygwalassin). Bioinformatic analysis of the requisite halogenase and dimethylallyltransferase revealed numerous other putative Trp-modified lasso peptides that remain uncharacterized. We anticipate that the Trp-centric strategy reported herein may be useful in discovering ancillary modifications for other RiPP classes and, more generally, guide the functional prediction of enzymes that act on specific amino acids.

Progesterone receptor membrane component 1 (PGRMC1) binds heme via a surface-exposed site and displays some structural resemblance to cytochrome b5 despite their different functions. In the case of PGRMC1, it is the protein interaction with drug-metabolizing cytochrome P450s and the epidermal growth factor receptor that has garnered the most attention. These interactions are thought to result in a compromised ability to metabolize common chemotherapy agents and to enhance cancer cell proliferation. X-ray crystallography and immunoprecipitation data have suggested that heme-mediated PGRMC1 dimers are important for facilitating these interactions. However, more recent studies have called into question the requirement of heme binding for PGRMC1 dimerization. Our study employs spectroscopic and computational methods to probe and define heme binding and its impact on PGRMC1 dimerization. Fluorescence, electron paramagnetic resonance and circular dichroism spectroscopies confirm heme binding to apo-PGRMC1 and were used to demonstrate the stabilizing effect of heme on the wild-type protein. We also utilized variants (C129S and Y113F) to precisely define the contributions of disulfide bonds and direct heme coordination to PGRMC1 dimerization. Understanding the key factors involved in these processes has important implications for downstream protein–protein interactions that may influence the metabolism of chemotherapeutic agents. This work opens avenues for deeper exploration into the physiological significance of the truncated-PGRMC1 model and developing design principles for potential therapeutics to target PGRMC1 dimerization and downstream interactions.

Telomeres are specialized structures, found at the ends of linear chromosomes in eukaryotic cells, that play a crucial role in maintaining the stability and integrity of genomes. They are composed of repetitive DNA sequences, ssDNA overhangs, and several associated proteins. The length of telomeres is linked to cellular aging in humans, and deficiencies in their maintenance are associated with various diseases. Key structural motifs at the telomeres serve to protect vulnerable chromosomal ends. Telomeric DNA also has the ability to form diverse complex DNA higher-order structures, including T-loops, D-loops, R-loops, G-loops, G-quadruplexes, and i-motifs, in the complementary C-rich strand. While many essential proteins at telomeres have been identified, the intricacies of their interactions and structural details are still not fully understood. This Perspective highlights recent advancements in comprehending the structures associated with human telomeres. It emphasizes the significance of telomeres, explores various telomeric structural motifs, and delves into the structural biology surrounding telomeres and telomerase. Furthermore, telomeric loops, their topologies, and the associated proteins that contribute to the safeguarding of telomeres are discussed.

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of β-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5′-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5′-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and β subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with β-l-lysine or d,l-lysine. Ultraviolet–visible (UV–vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg²⁺ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess β-l-lysine. Using a coupled UV–visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.