Biochemistry Biochemistry最新文献

Cyclic tetrapeptides (CTPs) are a diverse class of natural products with a broad range of biological activities. However, they are challenging to synthesize due to the ring strain associated with their small ring size. While chemical methods have been developed to access CTPs, they generally require the presence of certain amino acids, limiting their substrate scopes. Herein, we report the first bioinformatics-guided discovery of a thioesterase from a cryptic biosynthetic gene cluster for peptide cyclization. Specifically, we hypothesized that predicted penicillin-binding type thioesterases (PBP-TEs) from cryptic nonribosomal peptide synthetase (NRPS) gene clusters containing four adenylation domains would catalyze tetrapeptide cyclization. We found that one of the predicted PBP-TEs, WP516, efficiently cyclizes a wide variety of tetrapeptide substrates. To date, it is only the second stand-alone enzyme capable of efficiently cyclizing tetrapeptides, and its substrate scope greatly surpasses that of the only other reported tetrapeptide cyclase Ulm16. AlphaFold modeling, covalent docking, molecular dynamics, and mutational analyses were used to rationalize the broad substrate scope of WP516. Overall, the bioinformatics-guided workflow outlined in this paper and the discovery of WP516 represent promising tools for the biocatalytic production of head-to-tail CTPs, as well as a more general strategy for the discovery of enzymes for peptide cyclization.

Sweet proteins trigger sweet taste perception through interactions with the human T1R2/R3 sweet taste receptor. To date, relatively few proteins have been identified as causing sweet taste perception, and the four most studied proteins: monellin, brazzein, thaumatin, and honey truffle active component (HT-AC), have minimal sequence homology or structural similarities aside from positively charged surface sites. Sweet taste perception has also been found to be readily perturbed by minor changes in the protein structure, such as natural isoforms inherent to heterologous expression of the protein, and synthetic amino acid substitutions. This study uses ab initio rigid-body docking to predict the interactions of known sweet proteins and variants with a recently resolved cryo-EM structure of the T1R2/R3 sweet taste receptor, incorporating comparative analyses between apo-, holo-, and a potentially transient conformation of the receptor. HT-AC mediated activation of the sweet taste receptor is confirmed by in vitro cell-based assays, and results from in silico docking of various sweet proteins are used to derive additional insights regarding sweet taste perception. Perturbations of HT-AC due to naturally occurring post-translational modifications and synthetic modifications are evaluated using in vitro and in silico methods to determine robustness of the interaction between T1R2/R3 and sweet proteins with primary focuses on HT-AC.

Ubiquitination, a post-translational modification, regulates numerous cellular processes by attaching ubiquitin molecules to the target proteins, thereby altering their cellular levels and functions. A central aspect of ubiquitin-mediated signaling is the formation of different types of polyubiquitin (polyUb) chains, which can be either homotypic or heterotypic, generating a variety of cellular signals that activate distinct downstream pathways. Homotypic chains are linked via the same lysine on each molecule, whereas heterotypic chains are conjugated via multiple lysines. The synthesis of these diverse polyubiquitin chains is driven by interactions between substrate-conjugated ubiquitin molecules, ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). However, the molecular details of these interactions and how they govern the synthesis of different homotypic and heterotypic chains remain poorly understood. The E2 enzyme E2-25K preferentially extends K48-linked polyubiquitin chains on K63-linked template polyubiquitin chains, creating branched K48/K63 chains. In this study, we investigated the role of dynamic interactions between E2-25K and the K63-linked diubiquitin substrate to assess the molecular mechanism of branched-chain assembly. Our data reveal that E2-25K shows no preference for binding to the proximal or distal ubiquitin of a template chain. However, binding to the distal unit activates the complex; binding to the proximal unit does not. This study highlights the critical role of stereospecificity in enzyme/substrate interactions for branched ubiquitin chain synthesis and provides insights into the mechanisms of ubiquitin signaling.

RNA-binding proteins (RBPs) are essential regulators of posttranscriptional gene expression, influencing mRNA processing, translation, and stability. Defining their binding sites on RNA is key to understanding how they assemble into functional ribonucleoprotein (RNP) complexes, but existing footprinting and cross-linking approaches often yield low signal-to-noise, variable efficiency, or require highly purified complexes. To address these limitations, we developed Tethered Micrococcal Nuclease Mapping (TM-map), a sequencing-based strategy that determines the three-dimensional binding sites of RBPs on RNA in vitro. In TM-map, the RBP is fused to micrococcal nuclease (MNase), which upon Ca²⁺ activation cleaves proximal RNA regions, producing fragments whose 3′ termini report the spatial proximity of the fusion. We first validated TM-map using the bacteriophage MS2 coat protein bound to its cognate RNA stem-loop engineered into the Escherichia coli ribosome. Cleavage sites mapped proximal to the engineered stem-loop, confirming that tethered MNase reliably reports local protein-RNA proximity on the ribosome surface. We then applied TM-map to the Drosophila Fragile X Mental Retardation Protein (FMRP), a translational regulator with an unresolved ribosome-binding site. Both N- and C-terminal MNase-FMRP fusions produced reproducible cleavage clusters on the 18S rRNA localized to the body and head of the 40S subunit. The similar profiles suggest that FMRP’s termini are conformationally flexible and sample multiple orientations relative to the ribosome, consistent with a dynamic interaction rather than a fixed binding mode. TM-map thus provides a simple, proximity-based, and generalizable in vitro approach for visualizing RBP-RNA interactions within native RNP assemblies.

The Ras superfamily of small GTPases are challenging targets for therapeutic inhibition, partially due to a lack of pockets amenable to small molecule inhibition. Our previous work identified high-affinity cyclized peptide binders of Cdc42, a member of the Rho family of small GTPases, capable of inhibiting activity. To further optimize these Cdc42 inhibitors, we have engineered modifications to the best sequence available from the original maturation and screened the ability of these third-generation peptides to compete with Cdc42-effector interactions. Improvements in affinity were achieved by single amino acid substitutions at several residue positions. We present the structure of one of these nanomolar affinity, cyclized peptides in complex with Cdc42. The structure reveals that the peptide binds in a β-hairpin conformation to create an extension of the β-sheet of the GTPase Rossman fold, acting as a structural mimic of native Cdc42 effectors. We additionally elucidate the NMR structures of four unbound C-terminal alanine variants and employ both the bound and unbound structures to inform the rational design of substituted peptide inhibitors. Overall, this study expands our understanding of how Ras GTPases can be targeted, by demonstrating a rare example of an inhibitor binding contiguously with a surface of β-strand of the small G protein, which illustrates an innovative avenue for noncovalent therapeutic design.

Reader proteins that bind histone trimethyllysine (Kme3) have become active therapeutic targets in recent years. Binding of Kme3 in the conserved aromatic cages of these readers via cation-π interactions is a key contributor to these protein–protein interactions. We explored whether the replacement of one methyl group of Kme3 with an electron-withdrawing group (EWG) could improve binding by increasing the electrostatic component of the cation-π interaction. We examined these Kme3 analogs in binding studies with histone reader proteins, nuclear magnetic resonance (NMR) studies in a β-hairpin peptide model system, and computational studies to gain mechanistic insight into their binding. Kme3 analogs with both alkyl and benzyl substitution with EWGs were examined. We found that EWGs on the ligand differentially affected binding to a series of Kme3 readers but did not improve binding. This is likely due to sterics of the different analogs that preclude optimal cation-π interactions. The unique selectivity patterns of these ligands nonetheless offer promise toward selective inhibitor development within this protein class. NMR studies of the alkyl-substituted analogs in the β-hairpin model system show that EWGs strengthen the cation-π interaction enthalpically but with an entropic penalty. NMR studies demonstrate that the benzyl group makes direct contacts with Trp in the β-hairpin model, providing aromatic interactions in addition to cation-π interactions. Computational energy decomposition analysis confirms the more favorable electrostatic interactions compared to Kme3, but other factors counter the enhanced electrostatic component. This mechanistic investigation demonstrates how EWG-containing Kme3 analogs contribute to cation-π interactions and provide unique selectivity patterns relative to Kme3.

Quantum mechanical tunneling (QMT) is now recognized as a significant contributor to some enzyme catalyzed hydrogen transfer reactions. In this perspective, we examine recent theoretical and experimental advances that investigate when, and how, QMT contributes to enzyme catalysis. We highlight progress and challenges in computing the rate constants of reactions involving tunneling, including developments in semiclassical approaches and in nuclear-electronic orbital density functional theory. Case studies on flavoenzymes, ribonucleotide reductase, catechol O-methyl transferase and Morita–Baylis–Hillmanase illustrate how protein dynamics, vibrational gating and electrostatic effects apparently modulate barrier width and sustain tunneling-derived rate enhancements. We expect that continued integration of improved theoretical methods and dynamics-sensitive experiments will be essential to move QMT from a mechanistic phenomenon to a tunable design parameter in future enzyme engineering and rational catalyst development.

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections, for which there are few treatment options. The quorum sensing (QS) pathway governs many pathogenic behaviors that allow for P. aeruginosa to stage infections. Within the QS pathway, there is a key protein–protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa, also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. Here, we performed a fluorescence polarization screen of an FDA-approved drug library to identify molecules that bind in the active site of PqsE. Three molecules were identified, two of which showed inhibitory activity consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in a cellular enzyme assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein–protein interaction via binding in the PqsE active site.

Amyloid beta (Aβ) oligomers are thought to play an important role during development and progression of Alzheimer’s disease (AD). Previously, we determined the Aβ oligomer concentrations in various AD mouse models and in human brain tissues of former AD patients. Here, we investigate which proteins are part of these Aβ oligomers, apart from Aβ itself. Because several oligomer-associated proteins have been implicated in mechanisms leading to AD pathology, identification of the Aβ oligomer proteome may provide insights into the formation of Aβ oligomers in vivo and may reveal novel targets for disease-modifying therapeutic approaches. Here, we separated different native Aβ assemblies in brain homogenates of transgenic (tg) AD mice and human AD post mortem samples by density gradient centrifugation, then isolated Aβ-containing assemblies by co-immunoprecipitation. Mass spectrometry of immunoprecipitated proteins with label-free quantification (LFQ) showed significant changes between the proteomes of Aβ oligomers from tg AD mice and wildtype (wt) mice, confirming some proteins that have been expected to bind Aβ species, like ApoE and Clusterin, but also indicating novel, so far unknown, protein content of Aβ oligomers, such as the RabGAP Tbc1d10b. Some of the hereby identified proteins, like, for example, Clusterin, were also found to be enriched in Aβ oligomers from human AD brain tissue derived homogenates as compared to brain tissue from non-demented controls (NC). Others, such as Netrin-1, were specifically enriched in Aβ oligomers in AD compared to NC samples, but not in mouse samples.

Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles released by all cells. EVs carry nucleic acids, proteins, lipids, and metabolites in intercellular communication. As potential liquid-biopsy biomarkers and drug-delivery vehicles for diseases, however, isolating specific EV subpopulations remains challenging owing to their high heterogeneity in size, density, and surface protein markers. We utilized chemical biology tools to develop EV membrane-curvature probes, including MARCKS-ED and other peptides. These short, membrane-anchoring peptides detect the fluid membrane of EVs and report membrane curvature. Here, we review the peptide selection principles, bioorthogonal reaction engineering mechanisms, and applications of these EV peptide probes and discuss future directions, such as stimulus-responsive or artificial intelligence-assisted peptide probe design for on-demand EV capture and subpopulation.