Biochemistry Biochemistry最新文献

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective noncatechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and noncatechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, noncatechol agonists display G protein biased signaling at the D1R and have reduced or absent β-arrestin2 recruitment. A notable exception was compound (Cmpd) 19, a noncatechol agonist with full efficacy at both D1R-G protein and D1R-β-arrestin2 pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R-β-arrestin2 activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The β-arrestin2 super agonist, A-77636, showed statistically significant increases in D1R endocytosis. Moreover, β-arrestin2 recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential.

Enzyme-mediated site-specific protein modification is gaining attention in biopharmaceuticals due to its high specificity and mild conditions. Lipoic acid ligase A (LplA) has been widely studied for conjugating short-chain fatty acids to lysine residues, traditionally using LAP tags. Recent advances have enabled tag-free LplA modifications, expanding applications in antibody-drug conjugates (ADCs) and beyond. This study investigates the selective modification of Lys188 in trastuzumab by LplA. Spatial analysis and molecular modeling suggest that D151 and H189 facilitate nucleophilic attack and stabilize intermediates via electrostatic and π-cation interactions. These insights enhance our understanding of enzyme-driven site selectivity, guiding the rational design of antibody modifications. The findings support broader applications in ADC production, diagnostics, and next-generation biopharmaceuticals, emphasizing the role of local amino acid environments in enzymatic modifications.

Antimicrobial peptides (AMPs) have emerged as a promising solution to the escalating public health threat caused by multidrug-resistant bacteria. Although ongoing research efforts have established AMP's role in membrane permeabilization and leakage, the precise mechanisms driving these disruption patterns remain unclear. We leverage molecular dynamics (MD) simulations enhanced by membrane mimetic (HMMM) to systematically investigate how the physiochemical properties of magainin (+3) and pexiganan (+9) affect their localization, insertion, curvature perturbation, and membrane binding ensemble. Building on existing microbiology, NMR, circular dichroism, and fluorescence data, our analysis reveals that the lipid makeup is a key determinant in the binding dynamics and structural conformation of AMPs. We find that phospholipid type is crucial for peptide localization, demonstrated through magainin's predominant interaction with lipid tails and pexiganan's with polar headgroups in POPC/POPS membranes. The membrane curvature changes induced by pexiganan relative to magainin suggest that AMPs with larger charges have more potential in modulating bilayer bending. These insights advance our understanding of AMP-membrane interactions at the molecular level, offering guidance for the design of targeted antimicrobial therapies.

The rise of antibiotic resistance poses a severe global threat, specifically due to the emergence of multiresistant bacteria (ESKAPE pathogens), which are responsible for countless deaths globally. Consequently, the development of novel antibiotics is in dire need. Targeting proteins essential to DNA replication is a promising pathway, making the β-sliding clamp (β-SC) an attractive target. Currently, there are no antibiotics on the market that target the β-SC. However, numerous compounds are being investigated to create an antibiotic with high potency against a broad range of bacterial species. Interestingly, most proposed compounds do not bind to the entire active site, which may reduce their potential as high-potency inhibitors. This is due to the active site residue Met at position 362, adopting a "closed" conformation, preventing inhibitors access into Subsite II of the active site. This study explored the effect of key residues on the plasticity of the β-SC active site using molecular dynamics and metadynamics simulations under different physiological states. Our results show that the Met gate exhibits flexibility and both open and closed states are thermodynamically and kinetically accessible.

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in poultry and is the leading cause of gastrointestinal infections in the United States. Similar to other Gram-negative bacteria, C. jejuni possesses an extracellular carbohydrate-based capsular polysaccharide (CPS) composed of repeating units of monosaccharides bound via glycosidic linkages. The gene cluster for serotype 1 (HS:1) of C. jejuni contains 13 different genes required for the production and presentation of the CPS. Each repeating unit within the HS:1 CPS structure contains a backbone of glycerol phosphate and d-galactose. Here, the enzyme HS1.11 was shown to catalyze the formation of CDP-(2R)-glycerol from MgCTP and l-glycerol-3-phosphate. HS1.09 was found to be a multidomain protein that catalyzes the polymerization of l-glycerol-3-phosphate and d-galactose using UDP-d-galactose and CDP-(2R)-glycerol as substrates. The domain of HS1.09 that extends from residues 286 to 703 was shown to catalyze the transfer of l-glycerol-P from CDP-glycerol to the hydroxyl group at C4 of the d-galactose moiety at the nonreducing end of the growing oligosaccharide. The transfer of d-galactose to the C2 hydroxyl group of the glycerol-phosphate moiety was shown to be catalyzed with retention of configuration by the domain of HS1.09 that extends from residues 704 to 1095. Primers as short as a single d-galactoside were accepted as initial substrates. Oligosaccharide products were isolated by ion exchange chromatography and identified by high-resolution ESI-mass spectrometry and NMR spectroscopy.

Ran is a small GTPase of the Ras superfamily that governs nucleocytoplasmic transport, including that of miR-126, a microRNA that supports the homeostasis and expansion of leukemia stem cells (LSCs). Ran binds to Exportin 5 to facilitate the transport of precursor (pre)-miR-126 across the nuclear membrane for its maturation. Our goal is to inhibit Ran to prevent transport of pre-miR-126 to the cytoplasm. Like other Ras family proteins, targeting Ran with small molecules is challenging due to its relatively flat surface and lack of binding cavities. Ran's activity is regulated by a long and disordered C-terminus that provides opportunities for identifying cryptic binding pockets to target. We used a combination of molecular dynamics simulations and experiments and uncovered the critical role of the ensemble of the C-terminal conformations that enable the transition of Ran from the GTP-bound "on state" to its GDP-bound "off-state". We also showed that the Ran C-terminus allosterically modulates the conformations of residues in the nucleotide binding site and in the functionally relevant Switch 1 and 2 regions. Through computational deep mutational scans and experiments, we identified four residue hotspots L182, Y197, D200, and L201 at the core-C-terminus interface and four residue mutations V27A, E70D, N122A, and N122Y that mediate the allosteric communication between the core and switch regions. This information paves the way for our next step in the design of novel allosteric modulators for Ran.

Phosphorylation is a reversible post-translational modification that can modulate protein function. For example, phosphorylation modifications of solute carrier family 12 (SLC12) proteins function as molecular switches that precisely regulate cation-chloride ion transport. Elucidating the phosphoregulatory mechanism of SLC12 at the carboxy-terminal domain (CTD) through structural determination approaches remains challenging due to the domain's disordered and flexible nature. In this study, molecular dynamics (MD) simulations and enhanced sampling techniques were employed to investigate the CTD phosphoregulatory mechanism of SLC12A6 (also known as KCC3). Atomistic MD and metadynamics simulations revealed that the dephosphorylation of residues T940 and T997 stabilizes CTD to a favorable state that "switches on" the solvent accessibility of the inward-facing pocket. Meanwhile, phosphorylation induces distinct orientations of the CTD, transitioning the dimer into another favorable state that "switches off" the solvent accessibility. The alteration of solvent accessibility in the inward-facing pocket influences the water and ion dynamics. Based on these findings, we propose a "knob switch" model to illustrate how CTD phosphorylation regulates ion transport in KCC3.

G protein-coupled receptors (GPCRs) remain a focal point of research due to their critical roles in cell signaling and their prominence as drug targets. However, directly linking drug efficacy to the receptor-mediated activation of specific intracellular transducers and the resulting physiological outcomes remains challenging. It is unclear whether the enhanced therapeutic window of certain drugs─defined as the dose range that provides effective therapy with minimal side effects─stems from their low intrinsic efficacy across all signaling pathways or ligand bias, wherein specific transducer subtypes are preferentially activated in a given cellular system compared to a reference ligand. Accurately predicting safer compounds, through either low intrinsic efficacy or ligand bias, would greatly advance drug development. While AI models hold promise for such predictions, the development of deep learning models capable of reliably forecasting GPCR ligands with defined bioactivities remains challenging, largely due to the limited availability of high-quality data. To address this, we pretrained a model on receptor sequences and ligand data sets across all class A GPCRs and then refined it to predict low-efficacy compounds or biased agonists for individual class A GPCRs. This was achieved using transfer learning and a neural network incorporating natural language processing of target sequences and receptor mutation effects on signaling. These two fine-tuned models─one for low-efficacy agonists and one for biased agonists─are available on demand for each class A GPCR and enable virtual screening of large chemical libraries, thereby facilitating the discovery of compounds with potentially improved safety profiles.

The homodimeric cyclooxygenase enzymes (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. COX-2 behaves as a conformational heterodimer in solution comprised of allosteric (E_allo) and catalytic (E_cat) subunits that function cooperatively. We previously utilized ¹⁹F-nuclear magnetic resonance spectroscopy (¹⁹F-NMR) to show that the cyclooxygenase active site entrances in a COX-2 homodimer construct exhibited composite tightened and relaxed states that are dependent upon the type of ligand bound. A third state, hypothesized to represent the alteration of a loop comprised of residues 120-129, was also detected in the presence of ligands that allosterically potentiate activity. We report here studies that couple the use of ¹⁹F-NMR with COX-2 heterodimer constructs to characterize states arising in the individual subunits. Glycine and proline substitutions at Ser-121 were introduced to examine how these mutations alter the 120-129 loop. In the presence of AA, the subunits exhibited asymmetry, with tightened and relaxed states observed in E_allo and E_cat, respectively. Allosteric ligand binding resulted in a shift to equivalent symmetrical states, with tightened states observed in the presence of the allosteric inhibitor flurbiprofen and relaxed states observed in the presence of the allosteric potentiator palmitic acid. The S121P substitution results in a shift to equivalent relaxed states, as well as an alteration of the 120-129 loop in the absence of bound ligand. We put forth a model linking the observed differential states arising from allosteric ligand binding with structural transitions across the dimer interface that govern the regulation of cyclooxygenase activity.

Phytochromes are red-light-sensitive biliprotein photoreceptors that control a variety of physiological processes in plants, fungi, and bacteria. Lately, greater attention has been paid to these photoreceptors due to their potential as fluorescent probes for deep-tissue microscopy. Such fluorescing phytochromes have been generated by multiple amino acid substitutions in weakly fluorescent wild-type (WT) proteins. Remarkably, the single substitution of conserved Tyr176 by His in cyanobacterial phytochrome Cph1 increases the fluorescence quantum yield from 2.4 to 14.5%. In this work, we studied this Y176H variant by crystallography, MAS NMR, resonance Raman spectroscopy, and ultrafast absorption spectroscopy complemented by theoretical methods. Two factors were identified to account for the strong fluorescence increase. First, the equilibrium between the photoactive and fluorescent substates of WT Cph1 was shown to shift entirely to the fluorescent substate in Y176H. Second, structural flexibility of the chromophore is drastically reduced and the photoisomerization barrier is raised, thereby increasing the excited-state lifetime. The most striking finding, however, is that Y176H includes the structural properties of both the dark-adapted Pr and the light-activated Pfr state. While the chromophore adopts the Pr-typical ZZZssa configuration, the tongue segment of the protein adopts a Pfr-typical α-helical structure. This implies that Tyr176 plays a key role in coupling chromophore photoisomerization to the sheet-to-helix transition of the tongue and the final Pfr structure. This conclusion extends to plant phytochromes, where the homologous substitution causes light-independent signaling activity akin to that of Pfr.