Protein Science最新文献

Cannabidiol (CBD), the second most abundant of the active compounds found in the Cannabis sativa plant, is nonaddictive and of increasing interest due to its potential therapeutic utilities. The actions of CBD are mediated by cannabinoid receptors and other targets, both G protein-coupled receptors (GPCRs) and non-GPCR proteins. It has recently been shown that CBD is not an orthosteric ligand of the adenosine A_2A receptor (A_2AR), but rather a negative allosteric modulator. Because of the multitude non-conserved allosteric pockets in GPCRs, the binding mode of CBD to A_2AR remains unknown. To fill this knowledge gap, and due to the therapeutic relevance of CBD and A_2AR, we have used metadynamics simulations and site-directed mutagenesis to identify the binding mode of CBD. These theoretical and experimental results show that the allosteric binding site of CBD is along the ligand binding pathway of adenosine to A_2AR, near the orthosteric binding site.

In this study, we explored the design of linear D-tripeptides tailored to bind specific cavities of Gadd45β, chosen as a model protein target. To identify peptides that selectively interact with predicted binding sites, we combined computational modeling with biophysical experiments. Gadd45β was selected since it has emerged as a promising therapeutic target involved in multiple disease pathways, including cancer and inflammation. Computational analysis was first employed to characterize the structural features and potential binding sites of Gadd45β. Guided by these insights, linear D-tripeptides were designed and optimized for specific interactions with the target surface. The resulting candidates were subsequently assessed through a series of biophysical assays to evaluate their binding affinity, selectivity, and potential therapeutic activity. Complementary computational simulations were employed to gain atomistic insight into the dynamics of peptide-protein recognition. This integrated computational-experimental strategy led to the identification of two D-tripeptides, RYR and VWR, that bind Gadd45β at a biologically relevant site, illustrating a general framework for early-stage peptide ligand discovery.

IM30, the inner membrane-associated protein of 30 kDa (also known as Vipp1) is essential for thylakoid membrane biogenesis and/or maintenance in chloroplasts and cyanobacteria. IM30 and its bacterial homolog PspA belong to the ESCRT-III superfamily, proteins previously thought to be restricted to eukaryotes and archaea. Despite low sequence similarity, IM30 shares key structural and functional features with eukaryotic ESCRT-IIIs, including a conserved α1-α2 helical hairpin core and the ability to form oligomeric barrel or rod assemblies that mediate membrane remodeling. Using IM30 variants, we now show that membrane binding of IM30 is driven by electrostatic interactions between the positively charged α1-α3 helical hairpin and negatively charged lipid surfaces, paralleling the role of charged helical regions in some eukaryotic ESCRT-IIIs. This likely is followed by lateral assembly of IM30 into higher-order barrel or rod structures on the membrane. Once assembled, α0 helices within these oligomers engage and stabilize internalized membrane tubules, mirroring membrane interaction strategies of eukaryotic ESCRT-IIIs, which use both N-terminal sequence motifs and charged residues on α1/α2. Thus, our findings demonstrate a conserved membrane binding and remodeling mechanism across the ESCRT-III superfamily, underscoring an evolutionary link in membrane dynamics between pro- and eukaryotes.

The cytoplasm of a cell is inherently crowded with diverse biological macromolecules of varying sizes, which significantly influence thermodynamics and kinetics of essential processes such as protein folding, molecular association, and enzymatic reactions. Similarly, human eye lens is densely packed with crystallin proteins of different sizes, including multimers, oligomers and monomers, creating a highly crowded and concentrated environment. In this study, we explored the effects of poly ethylene glycol (PEG) of varying molecular weights and sizes (PEG-400, PEG-2k, PEG-6k, PEG-35k, and PEG-100k) on stability of HγDC. A uniform crowder concentration of 100 gL^-1 was maintained in all cases to mimic lens-like crowding conditions. Unfolding experiments were performed at 5 μM HγDC in 50 mM phosphate buffer (pH 7.4) using CD spectroscopy in the absence and presence of the crowders, monitoring ellipticity at 218 nm at a scan rate of 1 K/min. Our results revealed that crowders facilitate the thermal unfolding of HγDC, with the destabilizing effect increasing as the crowder size increases. Thermodynamic analysis showed that small crowders destabilize the protein primarily through enthalpic interactions, counterbalanced to some extent by entropic stabilization ( ${∆∆}^{\#}G={∆∆}^{\#}H-T∆{∆}^{\#}S$ ). In contrast, larger crowders (PEG-35k, PEG-100k) destabilize the protein almost entirely through entropic contributions, contradicting classical excluded volume theory. To rationalize these findings, we employed the associated water stabilization mechanism hypothesis, highlighting the role of associated water. Overall, our study emphasizes pivotal role of biological water in modulating protein stability, providing mechanistic insight into size-dependent crowding effects in human eye lens.

Tubulin detyrosination is an important α-tubulin specific posttranslational modification which has been implicated in various disorders including neurodegeneration and cancer. As such, the enzymes involved in the generation of this modification emerged as promising therapeutic targets. Previous studies have identified the members of the vasohibin family, VASH1 and VASH2, as the first class of enzymes involved in the generation of detyrosination. Recently, we have discovered Tubulin MetalloCarboxyPeptidase 1 (TMCP1) as the second class of enzymes catalyzing this modification. Here we describe the development of a highly sensitive FRET-based enzymatic assay to study and monitor the activity of TMCP1 and VASH2. The originality of this assay lies in the use of 3-nitrotyrosine as a quencher, which not only restores fluorescence upon cleavage but also closely mimics the natural tyrosine substrate, ensuring optimal enzyme recognition. The selected fluorogenic substrate, named FS2, exhibited strong quenching efficiency and a high signal-to-noise ratio, allowing for real-time kinetic monitoring of TMCP1 and VASH2 activity. Enzyme kinetics, competition assays, and metal ion dependency studies confirmed the assay's specificity, robustness, and physiological relevance. This optimized assay provides a powerful and reliable tool for the future identification and characterization of inhibitors of α-tubulin detyrosination.

The SARS-CoV-2 (spike protein is the primary target for vaccine design, with immunogens typically engineered to enhance stability by introducing proline mutations (2P) and mutating or deleting the furin cleavage site (FCS). While these modifications improve structural integrity, studies suggest that furin cleavage can play a functional role in spike protein dynamics, potentially enhancing ACE2 receptor binding. However, the impact of this cleavage on the unbound form of the spike protein remains unclear. In this study, we use extensive all-atom molecular dynamics simulations to compare the structural and dynamic properties of Cleaved and Uncleaved spike proteins in their prefusion, unbound state. Our results show that furin cleavage significantly alters allosteric communication within the protein, increasing correlated motions within the receptor binding domain (RBD) and N-terminal domain (NTD), which may facilitate receptor engagement. Principal component analysis reveals that the Cleaved and Uncleaved spike proteins sample distinct conformational landscapes, with Cleaved systems settling into stable basins more rapidly. In the receptor binding motif accessible conformation, the Cleaved spike samples two distinct tilt states-an inward (toward Closed-like) and an outward (toward Open-like) orientation-suggesting dynamic tuning between immune evasion and receptor accessibility. Additionally, furin cleavage primes the S2 subunit by expanding the base of the central helix, potentially influencing the transition to the post-fusion state. Glycan clustering patterns further suggest an adaptive structural response to cleavage, particularly in the NTD and RBD regions. These findings highlight the potential functional consequences of FCS deletion in immunogen design and underscore the importance of considering the native cleavage state in vaccine and therapeutic development.

The 16S microbial community profiling of a metagenomics library from geothermal spring at Lisvori (Lesvos island, Greece) enabled the identification of a putative sequence exhibiting 95% identity to the γ-type carbonic anhydrase (γ-CA) from Caloramator australicus (γ-CaCA). The sequence of γ-CaCA was amplified by PCR, cloned, and expressed in E. coli. Activity assays showed that γ-CaCA possesses very low, but detectable, anhydrase activity, while exhibiting no measurable esterase activity. Differential scanning fluorimetry (DSF) revealed that the enzyme shows high thermal stability with a melting temperature (T_m) approximately 65-75°C in the pH range between 5.5 and 9.0. The structure of γ-CaCA was determined by X-ray crystallography at 1.11 Å resolution, the highest resolution reported so far for a γ-CA. The enzyme was crystallized as a trimer in the crystallographic asymmetric unit and contains three zinc-binding sites, one at each interface of neighboring subunits of the trimer. Structure-based rational design enabled the design and creation of a mutant enzyme (γ-CaCAmut) which possessed a heptapeptide insertion at the active-site loop and two-point mutations. Kinetic analysis demonstrated that γ-CaCAmut was successfully converted into a catalytically active esterase indicating successful activity gain through structure-guided engineering. The thermostability of γ-CaCAmut was significantly increased, aligning with the thermostability typically observed in hyperthermostable enzymes. X-ray crystallographic analysis of the γ-CaCAmut structure at 2.1 Å resolution, provided detailed structural insights into how the mutations impact the overall enzyme structure, function, and thermostability. These findings provide valuable structural and functional insights into γ-CAs and demonstrate a strategy for converting an inactive enzyme into a catalytically active form through rational design.

Dysregulation of the phosphatase SHP2 is implicated in various diseases, including congenital disorders and cancer. SHP2 contains two phosphotyrosine-recognition domains (N-SH2 and C-SH2) and a protein tyrosine phosphatase (PTP) domain. The N-SH2 domain is critical for SHP2 regulation. In the auto-inhibited state, it binds to the PTP domain and blocks the active site, but phosphoprotein engagement destabilizes the N-SH2/PTP domain interaction, thereby exposing the active site. Many disease mutations in SHP2 are at the N-SH2/PTP interface, and they hyperactivate SHP2 by disrupting auto-inhibitory interactions. The activating E139D mutation represents an exception to this mechanism, as it resides in the C-SH2 domain and makes minimal interactions in auto-inhibited and active state crystal structures. In this study, using AlphaFold2 modeling and molecular dynamics simulations, we identify an alternative active conformation of SHP2, in which Glu139 interacts with Arg4 and Arg5 on the N-SH2 domain to stabilize a novel N-SH2/C-SH2 interface. Using double mutant cycles, we show that this active state is further stabilized by the E139D mutation. Finally, we demonstrate that the E139D mutation enforces an active conformation with distinct phosphoprotein binding preferences from canonical hyperactive mutants. Thus, our study reveals a novel mechanism for SHP2 dysregulation.

Lactate dehydrogenase (LDH) is a key enzyme in cancer metabolism, with isoforms LDH5 and LDH1 supporting glycolysis and oxidative lactate metabolism, respectively. While the development of competitive LDH inhibitors has faced diverse challenges, allosteric strategies targeting LDH tetramerization have recently attracted increasing attention. To further explore this alternative, we investigated the factors influencing LDH tetramerization and enzymatic activity using a truncated form of human LDH-B (LDHBtr), which was reported to exist predominantly as a dimer. Unexpectedly, LDHBtr exhibited measurable activity at high concentrations, correlating with increased protein stability and a structural transition to the tetrameric form. Preincubation with NADH further enhanced LDHBtr activity, stability, and self-association, consistent with cofactor-promoted tetramer assembly. Crystallographic studies confirmed the tetrameric structure of LDHBtr bound to NADH. Furthermore, reported LDH allosteric inhibitors, including cGmC9 and fluoxetine, preferentially inhibited LDHBtr compared to the native LDHB, by preventing tetramer formation. Overall, this work highlights the central role of tetramerization in regulating LDH activity, and the therapeutic potential of targeting this process. It also establishes LDHBtr as a valuable tool for screening tetramerization disruptors, paving the way for next-generation LDH inhibitors to target cancer metabolism.

Many proteins harbor covalent intramolecular bonds that enhance their stability and resistance to thermal, mechanical, and proteolytic insults. Intramolecular isopeptide bonds represent one such covalent interaction, yet their distribution across protein domains and organisms has been largely unexplored. Here, we sought to address this by employing a large-scale prediction of intramolecular isopeptide bonds in the AlphaFold database using the structural template-based software Isopeptor. Our findings reveal an extensive phyletic distribution in bacterial and archaeal surface proteins resembling fibrillar adhesins and pilins. All identified intramolecular isopeptide bonds are found in two structurally distinct folds, CnaA-like or CnaB-like, from a relatively small set of related Pfam families, including 10 novel families that we predict to contain intramolecular isopeptide bonds. One CnaA-like domain of unknown function, DUF11 (renamed here to "CLIPPER") is broadly distributed in cell-surface proteins from Gram-positive bacteria, Gram-negative bacteria, and archaea, and is structurally and biophysically characterized in this work. Using x-ray crystallography, we resolve a CLIPPER domain from a Gram-negative fibrillar adhesin that contains an intramolecular isopeptide bond and further demonstrate that it imparts thermostability and resistance to proteolysis. Our findings demonstrate the extensive distribution of intramolecular isopeptide bond-containing protein domains in nature and structurally resolve the previously cryptic CLIPPER domain.