Biochemistry Biochemistry最新文献

Cytochrome c nitrite reductase (NrfA) is a pentaheme enzyme capable of the six-electron reduction of nitrite to ammonia, which is a key step in the nitrogen cycle. All NrfA enzymes appear to have a branched set of two heme-based pathways for electron transfer to a conserved active site, and until recently, NrfA enzymes from a variety of microorganisms were considered to possess a homodimeric structure; yet, recent efforts have shown that in solution, purified Geobacter lovleyi (Gl) NrfA is a monomer. Direct protein electrochemistry has been used in the past to characterize the dimeric NrfAs from Escherichia coli and Shewanella oneidensis, revealing features of maximal activity as a function of nitrite concentration, and redox poise, both of which were interpreted in terms of the dimeric structure providing multiple redox equivalents. Here, we examine Gl NrfA using protein film electrochemistry and find that all of the features that were associated with the dimeric enzymes are also found in the monomeric enzyme. Further, we probe the contribution of specific heme environments through investigation of two His to Met heme ligand mutants, each along a different branch of the electron transfer network, which demonstrates that each path is likely essential to support native-like catalysis.

Bacterial microcompartments (BMCs) are nanometer-scale organelles with a protein-based shell that serve to colocalize and encapsulate metabolic enzymes. They may provide a range of benefits to improve pathway catalysis, including substrate channeling and selective permeability. Several groups are working toward using BMC shells as a platform for enhancing engineered metabolic pathways. The microcompartment shell of Haliangium ochraceum (HO) has emerged as a versatile and modular shell system that can be expressed and assembled outside its native host and with non-native cargo. Further, the HO shell has been modified to use the engineered protein conjugation system SpyCatcher-SpyTag for non-native cargo loading. Here, we used a model enzyme, triose phosphate isomerase (Tpi), to study non-native cargo loading into four HO shell variants and begin to understand maximal shell loading levels. We also measured activity of Tpi encapsulated in the HO shell variants and found that activity was determined by the amount of cargo loaded and was not strongly impacted by the predicted permeability of the shell variant to large molecules. All shell variants tested could be used to generate active, Tpi-loaded versions, but the simplest variants assembled most robustly. We propose that the simple variant is the most promising for continued development as a metabolic engineering platform.

Abl1, a nonreceptor tyrosine kinase closely related to Src kinase, regulates critical cellular processes like proliferation, differentiation, cytoskeletal dynamics, and response to environmental cues through phosphorylation-driven activation. Dysregulation places it centrally in the oncogenic pathway leading to blood cancers. making it an ideal drug target for small molecule inhibitors. We sought to understand the underlying mechanism of the phosphoryl-transfer step from the ATP molecule to the substrate tyrosine, as carried out by the Abl1 enzyme. By calculating free energy profiles for the reaction using the empirical valence bond representation of the reacting fragments paired with molecular dynamics and free energy perturbation calculations, a combination of several plausible reaction pathways, ATP conformations, and the number of magnesium ion cofactors have been investigated. For the best-catalyzed pathway, which proceeds through a dissociative mechanism with a single magnesium ion situated in Site I, a close agreement was reached with the experimentally determined catalytic rates. We conclude that the catalytic mechanism in Abl1 requires one magnesium ion for efficient catalysis, unlike other kinases, where two ions are utilized. A better overall understanding of the phosphoryl-transfer reactions in Abl1 can be used for type-I inhibitor development and generally contributes to a comprehensive overview of the mechanism for ATP-driven reactions.

Biased signaling has transformed pharmacology by revealing that receptors, particularly G protein-coupled receptors (GPCRs), can activate specific intracellular pathways selectively rather than uniformly. This discovery enables the development of targeted therapeutics that minimize side effects by precisely modulating receptor activity. Functionally selective ligands, which preferentially activate distinct signaling branches, have become essential tools for exploring receptor mechanisms and uncovering the complexities of GPCR signaling. These ligands help clarify receptor function in various physiological and pathological contexts, offering profound implications for therapeutic innovation. GPCRs, which mediate a wide range of cellular responses through coupling to G proteins and arrestins, are key pharmacological targets, with nearly a third of FDA-approved drugs acting on them. Recent advancements in biosensor development, multiplex assay platforms, and deep mutational scanning methods are improving our ability to define GPCR signaling, allowing for a better understanding of biased signaling pathways.

Spastin is a microtubule-severing AAA+ ATPase that is highly expressed in neuronal cells and plays a crucial role in axonal growth, branching, and regeneration. This machine oligomerizes into hexamers in the presence of ATP and microtubule carboxy-terminal tails (CTTs). Conformational changes in spastin hexamers, powered by ATP hydrolysis, apply forces to the microtubule, ultimately leading to the severing of the filament. Mutations disrupt the normal function of spastin, impairing its ability to sever microtubules effectively and leading to abnormal microtubule dynamics in neurons characteristic of the set of neurodegenerative disorders called hereditary spastic paraplegias (HSP). Experimental studies have identified the HSP-related R591S (Drosophila melanogaster numbering) mutation as playing a crucial role in spastin. Given its significant role in HSP, we employed a combination of molecular dynamics simulations with machine learning and graph network-based approaches to identify and quantify the perturbations caused by the R591S HSP mutation on spastin's dynamics and allostery with functional implications. We found that the functional hexamer, upon HSP-related mutation, loses the ability to execute the primary motion associated with the severing action. The study of allosteric changes upon the mutation showed that the regions that are most perturbed are those involved in the formation of the interprotomer contacts. The mutation induces rigidity in the allosteric networks of the motor, making it more likely to experience loss of function as applied perturbations would not be easily dissipated by passing through a variety of alternative paths as in the wild-type (WT) spastin.

Aptamers bind to their targets with exceptional affinity and specificity. However, their intracellular application is hampered by the lack of knowledge about the effect of the cellular milieu on the RNA structure/stability. In this study, cellular crowding was mimicked using polyethylene glycol (PEG), and the crucial role of Mg²⁺ ions in stabilizing the structure of an RNA aptamer was investigated. Increasing the concentration of Mg²⁺ or PEG increased the thermal stability of the aptamer. The crowding effect lowered the requirement of the Mg²⁺ ion to form the binding-competent conformer of the aptamer. This suggests that crowding and other factors may compensate for a lower concentration of Mg²⁺ for proper folding of aptamers inside cells. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing permitted residue-level analysis of the aptamer. Mg²⁺ and/or PEG were shown to be involved in increasing the rigidity or flexibility of different regions of the aptamer. Fluorescence correlation spectroscopy showed a significantly low hydrodynamic radius (R_H) in the presence of molecular crowders and Mg²⁺ ions. We believe that the decreased water activity due to crowding may be responsible for reduced R_H. Our results show that in a crowded environment, the RNA aptamer was exposed to conformers that were not available to it in simple buffer solutions or solely in the presence of lower concentrations of Mg²⁺.

Several human transcription factors (TFs) have been reported to directly bind RNA through noncanonical RNA-binding domains; however, most of these TFs remain to be further validated as bona fide RNA-binding proteins (RBPs). Our systematic analysis of RBP discovery data sets reveals a varied set of candidate TF-RBPs that encompass most TF families. These candidate RBPs include members of the GATA family that are essential factors in embryonic development. Investigation of the RNA-binding features of GATA1, a major hematopoietic TF, reveals robust sequence independent binding to RNAs in vitro. Moreover, RNA binding by GATA1 is competitive with DNA binding, which occurs through a shared binding surface spanning the DNA-binding domain and arginine-rich motif (ARM)-like domain. We show that the ARM-like domain contributes substantially to high-affinity DNA binding and electrostatically to plastic RNA recognition, suggesting that the separable RNA-binding domain assigned to the ARM-domain in GATA1 is an oversimplification of a more complex recognition network. These biochemical data demonstrate a unified integration of DNA- and RNA-binding surfaces within GATA1, whereby the ARM-like domain provides an electrostatic surface for RNA binding but does not fully dominate GATA1-RNA interactions, which may also apply to other TF-RBPs. This competitive DNA/RNA binding activity using overlapping nucleic acid binding regions points to the possibility of RNA-mediated regulation of the GATA1 function during hematopoiesis. Our study highlights the multifunctionality of DNA-binding domains in RNA recognition and supports the need for robust characterization of predicted noncanonical RNA-binding domains such as ARM-like domains.

The fine-tuning of transcription factor DNA-binding activity is often governed by transient intramolecular interactions between the transactivation domain and the DNA-binding domain. An example of such interaction is found in the transcription factor ATF4, a central regulator of the Integrated Stress Response. In ATF4, dynamic coupling between the transactivation domain and the basic-leucine zipper (bZip) domain modulates the phosphorylation levels of the disordered transactivation domain by casein kinase 2. However, the structural and molecular basis of these interdomain interactions remains poorly understood. This study focuses on a secondary basic motif at the C-terminus of ATF4, which is shared exclusively with its closest paralogue, ATF5. Through a combination of solution NMR spectroscopy, fluorescence polarization assays, and long-timescale molecular simulations, we demonstrate that this secondary basic motif is the primary driver of interdomain coupling between the transactivation and bZip domains of ATF4. Moreover, this motif enhances ATF4's DNA-binding specificity via interaction with the transactivation domain while also potentially facilitating rapid DNA scanning. Our findings reveal the pivotal role of a conserved motif in establishing disorder-mediated interactions that critically modulate ATF4's DNA-binding activity.

Cell-penetrating peptides (CPPs) are known for their effective intracellular transport of bioactives such as therapeutic proteins, peptides, nucleic acid, and small molecule drugs. However, the excessive cationic charges that promote their membrane permeability result in nonselective delivery and cellular toxicity. In this study, we report a decamer cell-penetrating peptidomimetic, Hkd, designed to selectively deliver anticancer drugs into tumor cells in response to the acidic microenvironment. The pH-sensitive histidine (H) imidazole side chain undergoes protonation in acidic environments, facilitating membrane permeability. The rigid cyclic dipeptide (CDP) core (kd) of Hkd has multiple hydrogen bond donor and acceptor sites, enabling selective interaction-driven cellular uptake. Pharmacokinetic studies revealed the excellent serum stability of Hkd. Cellular uptake studies of Hkd showed improved uptake at a lower pH than physiological pH. Conjugation of Hkd to the anticancer drug camptothecin (Cpt) reduced nonselective drug transport to normal cells while effectively delivering the drug into cancerous cells at the tumor microenvironment pH and retaining the therapeutic potential of the drug. The systematic design of pH-sensitive peptidomimetics offers a viable method to overcome the challenges of stability and selectivity faced by traditional highly cationic CPPs, potentially expanding the application range of this delivery system.

The increase in antimicrobial resistance presents a major challenge in treating bacterial infections, underscoring the need for innovative drug discovery approaches and novel inhibitors. Bacterial RNA polymerase (RNAP) has emerged as a crucial target for antibiotic development due to its essential role in transcription. RNAP is a molecular motor, and its function relies heavily on the dynamic shifts between multiple conformational states. While biochemical and structural experimental methods offer crucial insights into static RNAP-drug interactions, they fall short in capturing the dynamics at a molecular level. By integrating experimental data with advanced computational techniques like Markov State Models (MSMs), Generalized Master Equation (GME) Models and other machine-learning models constructed from molecular dynamics (MD) simulations, researchers can elucidate novel cryptic pockets that open transiently for antibiotic compounds and gain a more nuanced and comprehensive understanding of RNAP-drug interactions. This integrated approach not only deepens our fundamental knowledge but also enables more targeted and efficient antibiotic design strategies. In this Perspective, we highlight how this synergy between experimental and computational methods has the potential to open new pathways for innovative drug design and combination therapies that may help turn the tide in the ongoing battle against antibiotic-resistant bacteria.