Biochemistry Biochemistry最新文献

Human guanylate binding proteins (hGBPs), which are large GTPases, are crucial for cell-autonomous immunity, including antiviral activity. hGBPs contain two domains: an N-terminal catalytic domain and a C-terminal helical domain. hGBP3 and its splice variant hGBP3ΔC have been shown to possess anti-influenza activity in lung epithelial cells. These two proteins have identical catalytic domains but different helical domains. It is unclear whether this difference affects GTPase activity or protein oligomerization. Using combined approaches, we show that both proteins hydrolyze GTP to GDP and further to GMP. However, they form different oligomers. hGBP3 exists as a hexamer in the free form, whereas hGBP3ΔC forms large oligomers, indicating that helical domain modifications of the splice variant result in distinct oligomers. Furthermore, unlike other homologues, neither protein changes its oligomeric state upon substrate binding or hydrolysis. Deleting the helical domain of hGBP3 (hGBP3^1-309) yields a monomer, suggesting that the helical domain promotes the hexamerization of hGBP3. We overexpressed hGBP3 and hGBP3ΔC to test their efficacy against HCV growth and found that hGBP3 inhibits HCV multiplication, while the splice variant has little effect. Our mutational studies on hGBP3 show that substrate hydrolysis, rather than substrate binding, is required for inhibiting HCV growth. This suggests that substrate hydrolysis generates a protein conformation essential for anti-HCV activity. Additionally, truncated hGBP3^1-309 does not exhibit anti-HCV activity. Altogether, these findings suggest that the helical domain of hGBP3 is crucial for reducing HCV growth through hexamer formation and that its variations result in different oligomers and antiviral activities.

Mutations in dynamin 2 (DNM2) have been associated with two distinct movement disorders: Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). Most of these mutations are clustered in the pleckstrin homology domain (PHD), which engages in intramolecular interactions that limit dynamin self-assembly and GTPase activation. CNM mutations interfere with these intramolecular interactions and suppress the formation of the autoinhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for phosphoinositide binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations causes either one disease or the other, despite their close proximity within the PHD sequence. We previously reported that at least one CMT-causing mutant, lacking residues ₅₅₅DEE₅₅₇ (ΔDEE), displays the same inability to undergo autoinhibition as observed in CNM-linked mutants. Here, we show that both the DNM2^ΔDEE and CNM-linked DNM2^A618T mutants form larger and more stable structures on the plasma membrane than that of wild-type DNM2 (DNM2^WT). However, DNM2^A618T forms cytoplasmic inclusions at concentrations lower than those of either DNM2^WT or DNM2^ΔDEE, suggesting that CNM-linked mutations confer more severe gain-of-function properties than the ΔDEE mutation.

The in vitro transcription (IVT) of messenger ribonucleic acid (mRNA) from the linearized deoxyribonucleic acid (DNA) template of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) was optimized for total mRNA yield and purity (by percent intact mRNA) utilizing machine learning in conjunction with automated, high-throughput liquid handling technology. An iterative Bayesian optimization approach successfully optimized 11 critical process parameters in 42 reactions across 5 experimental rounds. Once the optimized conditions were achieved, an automated, high-throughput screen was conducted to evaluate commercially available T7 RNA polymerases for rate and quality of mRNA production. Final conditions showed a 12% yield improvement and a 50% reduction in reaction time, while simultaneously significantly decreasing (up to 44% reduction) the use of expensive reagents. This novel platform offers a powerful new approach for optimizing IVT reactions for mRNA production.

The secondary plastoquinone (PQ) electron acceptor Q_B in photosystem II (PSII) undergoes a two-step photoreaction through electron transfer from the primary PQ electron acceptor Q_A, converting into plastoquinol (PQH₂). However, the detailed mechanism of the Q_B reactions remains elusive. Here, we investigated the reaction mechanism of Q_B in cyanobacterial PSII core complexes using two time-revolved infrared (TRIR) methods: dispersive-type TRIR spectroscopy and rapid-scan Fourier transform infrared spectroscopy. Upon the first flash, the ∼140 μs phase is attributed to electron transfer from Q_A^•- to Q_B, while the ∼2.2 and ∼440 ms phases are assigned to the binding of an internal PQ in a nearby cavity to the vacant Q_B site and an external PQ traveling to the Q_B site through channels, respectively, followed by immediate electron transfer. The resultant Q_B^•- is suggested to be in equilibrium with Q_BH^•, which is protonated at the distal oxygen. Upon the second flash, the ∼130 μs and ∼3.3 ms phases are attributed to electron transfer to Q_BH^• and the protonation of Q_B^•- followed by electron transfer, respectively, forming Q_BH^-, which then immediately accepts a proton from D1-H215 at the proximal oxygen to become Q_BH₂. The resultant D1-H215 anion is reprotonated in ∼22 ms via a pathway involving the bicarbonate ligand. The final ∼490 ms phase may reflect the release of PQH₂ and its replacement with PQ. The present results highlight the importance of time-resolved infrared spectroscopy in elucidating the mechanism of Q_B reactions in PSII.

Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr-cis/trans-Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, R¹CO-pSer-Pro-NHR², was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with ¹⁵N-labeled Pin1 were used to measure K_d for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain.

The kinetics of the interaction between Musashi-1 (MSI1) and RNA have been characterized using surface plasmon resonance biosensor analysis. Truncated variants of human MSI1 encompassing the two homologous RNA recognition motifs (RRM1 and RRM2) in tandem (aa 1-200), and the two RRMs in isolation (aa 1-103 and aa 104-200, respectively) were produced. The proteins were injected over sensor surfaces with immobilized RNA, varying in sequence and length, and with one or two RRM binding motifs. The interactions of the individual RRMs with all RNA variants were well described by a 1:1 interaction model. The interaction between the MSI1 variant encompassing both RRM motifs was bivalent and rapid for all RNA variants. Due to difficulties in fitting this complex data using standard procedures, we devised a new method to quantify the interactions. It revealed that two RRMs in tandem resulted in a significantly longer residence time than a single RRM. It also showed that RNA with double UAG binding motifs and potential hairpin structures forms less stable bivalent complexes with MSI1 than the single UAG motif containing linear RNA. Substituting the UAG binding motif with a CAG sequence resulted in a reduction of the affinity of the individual RRMs, but for MSI1, this reduction was strongly enhanced, demonstrating the importance of bivalency for specificity. This study has provided new insights into the interaction between MSI1 and RNA and an understanding of how individual domains contribute to the overall interaction. It provides an explanation for why many RNA-binding proteins contain dual RRMs.

Sensory rhodopsin II (SRII) is a prototype photosensor that binds the retinal Schiff base chromophore. Upon photoabsorption, SRII is transformed into the signaling state, where two long-lived photointermediates are known to contribute. One is the M intermediate containing the deprotonated 13-cis chromophore, and the other is the O intermediate that is believed to have the protonated all-trans chromophore. The chromophore in the O intermediate is also thought to have the atypical 15-syn (C═N cis) configuration about the Schiff base moiety. In this communication, we study SRII from Natronomonas pharaonis (NpSRII) using Raman spectroscopy and find that the retinal chromophore configuration in the O intermediate is the 13-cis, 15-anti (C═N trans), contrary to the conventional notion. This result points out the revision of the chromophore structural changes underlying the long-lived signaling state of SRII.

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ¹-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. N-propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH. This compound consists of a recognition module (glycine) that directs the inactivator to the active site and an alkyne warhead that reacts with the FAD after oxidative activation, leading to covalent modification of the FAD N5 atom. Here we report structural and kinetic data on analogs of N-propargylglycine with the goals of understanding the initial docking step of the inactivation mechanism and to test the allyl group as a warhead. The crystal structures of PRODH complexed with unreactive analogs in which N is replaced by S show how the recognition module mimics the substrate proline by forming ion pairs with conserved arginine and lysine residues. Further, the C atom adjacent to the alkyne warhead is optimally positioned for hydride transfer to the FAD, providing the structural basis for the first bond-breaking step of the inactivation mechanism. The structures also suggest new strategies for designing improved N-propargylglycine analogs. N-allylglycine, which consists of a glycine recognition module and allyl warhead, is shown to be a covalent inactivator; however, it is less efficient than N-propargylglycine in both enzyme inactivation and cellular assays. Crystal structures of the N-allylglycine-inactivated enzyme are consistent with covalent modification of the N5 by propanal.

Fungal ribosomally synthesized and post-translationally modified peptides (RiPPs) are a vital class of natural products known for their biological activities including anticancer, antitubulin, antinematode, and immunosuppressant properties. These bioactive fungal RiPPs play key roles in chemical ecology and have a significant therapeutic potential. Their structural diversity, which arises from intricate post-translational modifications of precursor peptides, is particularly remarkable. Despite their biological and ecological importance, the discovery of fungal RiPPs has been historically challenging and only a limited number have been identified. To date, known fungal RiPPs are primarily grouped into three groups: cycloamanides and borosins from basidiomycetes and dikaritins from ascomycetes. Recent advancements in bioinformatics have revealed the vast untapped potential of fungi to produce RiPPs, offering new opportunities for their discovery. This review highlights recent progress in fungal RiPP biosynthesis and genome-guided discovery strategies. We propose that combining the knowledge of fungal RiPP biosynthetic pathways with advanced gene-editing technologies and bioinformatic tools will significantly accelerate the discovery of novel bioactive fungal RiPPs.

Drosophila Pins (and its mammalian homologue LGN) play a crucial role in the process of asymmetric cell division (ACD). Extensive research has established that Pins/LGN functions as a conformational switch primarily through intramolecular interactions involving the N-terminal TPR repeats and the C-terminal GoLoco (GL) motifs. The GL motifs served as binding sites for the α subunit of the trimeric G protein (Gα), which facilitates the release of the autoinhibited conformation of Pins/LGN. While LGN has been observed to specifically bind to Gα_i·GDP, Pins has been found to associate with both Drosophila Gα_i (dGα_i) and Gα_o (dGα_o) isoforms. Moreover, dGα_o was reported to be able to bind Pins in both the GDP- and GTP-bound forms. However, the precise mechanism underlying the influence of dGα_o on the conformational states of Pins remains unclear, despite extensive investigations into the Gα_i·GDP-mediated regulatory conformational changes in LGN/Pins. In this study, we conducted a comprehensive characterization of the interactions between Pins-GL motifs and dGα_o in both GDP- and GTP-loaded forms. Our findings reveal that Pins-GL specifically binds to GDP-loaded dGα_o. Through biochemical characterization, we determined that the intramolecular interactions of Pins primarily involve the entire TPR domain and the GL23 motifs. Additionally, we observed that Pins can simultaneously bind three molecules of dGα_o·GDP, leading to a partial opening of the autoinhibited conformation. Furthermore, our study presents evidence contrasting with previous observations indicating the absence of binding between dGα_i and Pins-GLs, thus implying the pivotal role of dGα_o as the principal participant in the ACD pathway associated with Pins.