Biochemistry Biochemistry最新文献

Collagen embodies an intriguing paradox in protein biology. Despite being one of the most abundant protein superfamilies in vertebrates and having a seemingly simple structural organization, its biosynthesis is anything but straightforward. This apparent simplicity masks a complex and often contradictory biosynthetic landscape that poses significant challenges, particularly for newcomers to the field. Rather than following a linear or uniform pathway, collagen biosynthesis involves a coordinated series of tightly regulated steps, cotranslational post-translational modifications (PTMs), chain selection and registration, triple helix formation, and secretion, orchestrated by a specialized machinery, collectively termed the collagen molecular ensemble. This ensemble must overcome unconventional paradigms in protein biogenesis, rife with exceptions and unresolved questions. In this perspective, I examine underexplored aspects of the collagen biosynthetic machinery, spotlighting challenges in decoding the regulatory logic of PTMs, the spatial dynamics of trimer assembly, the functional consequences of chain registration, and the type-specific routes of secretion. By charting these uncertainties, I aim to challenge prevailing assumptions and invite interdisciplinary insight to help unravel the remaining mysteries of collagen biosynthesis.

G protein-coupled receptors (GPCRs) make up the largest receptor family in humans, which also constitute principal molecular targets for about 36% of approved drugs. Recent studies show that GPCRs can form heteromeric complexes with new molecular features. Little, however, is known about how human bitter taste receptors (T2Rs) form heteromeric receptors with other GPCRs. In this study, we combine biomolecular fluorescence complementation assays with methods for chemiluminescence imaging of cells, and find that β2-adrenergic receptor (β2AR) interacts with a subset of T2Rs, including T2R10, T2R14, T2R38, and T2R44, but selectively promotes cell membrane localization of only T2R14, T2R38, and T2R44. Furthermore, in silico modeling, coimmunoprecipitation, and immunofluorescence analysis indicate that β2AR utilizes distinct interfacial domains to interact with different T2Rs. And the β2AR-T2R14 interaction is selectively disrupted by a synthetic peptide corresponding to the transmembrane helix 4 of β2AR, which, however, does not block ligand-induced β2AR or T2R14 receptor internalization. Taken together, our findings demonstrate that β2AR employs different transmembrane helices to interact with and regulate special T2R subtypes. The insights obtained from this research may further our understanding of the β2AR-T2R interaction mechanisms and facilitate the development of new clinical drugs targeting β2AR-T2R complexes.

Endolysins are phage-encoded enzymes that cleave the peptidoglycan of host bacteria. These enzymes have gained considerable attention due to their ability to cause cell lysis, making them candidates as antibacterial agents. Most Pseudomonas aeruginosa genomes, including the common laboratory strains PAO1 and UCBPP-PA14, contain a cryptic prophage encoding a glycoside hydrolase family 19 endolysin (named PaGH19Lys in the present study). Family 19 glycoside hydrolases are known to target peptidoglycan and chitin-type substrates. PaGH19Lys was not active toward chitin but exhibited activity toward chloroform-treated Gram-negative bacteria, displaying ∼10,000-fold higher activity than hen egg white lysozyme. Analysis of products derived from PaGH19Lys activity toward purified P. aeruginosa peptidoglycan showed that the enzyme catalyzed hydrolysis of the β-1,4 linkage between N-acetylmuramic acid and N-acetyl-d-glucosamine, classifying the enzyme as a muramidase. Finally, the crystal structure of PaGH19Lys was determined and solved to 1.8 Å resolution. The structure of the enzyme showed a globular α-helical fold possessing a deep but relatively open catalytic cleft.

Members of the ATG8 family of ubiquitin-like proteins (Ubls) are covalently attached to phosphatidylethanolamine (PE) on nascent autophagosomal membranes, where they recruit cargo receptors and promote membrane expansion. Although the overall lipidation pathway is well established, the molecular details-particularly those involving the E2 enzyme ATG3-remain incompletely defined. Here, we uncover a previously unrecognized, noncovalent binding mode between the mammalian ATG8 protein GABARAP and the backside of ATG3's catalytic E2 domain. In crystals, an isopeptide-linked GABARAP∼ATG3 conjugate self-assembles into a helical filament via this backside interface, mirroring architectures observed for canonical Ub/Ubl∼E2 conjugates. The E2 backside-binding surface on GABARAP is topologically distinct from those of other Ub/Ubl proteins and overlaps the LC3-interacting region (LIR) motif-binding site. Solution NMR confirms this interaction, and targeted mutagenesis shows that disrupting the interface impairs PE conjugation. Complementary NMR and AlphaFold modeling of apo ATG3 reveal an intramolecular contact between a segment of its flexible region (FR) and the catalytic core that suppresses conjugation. Together, these findings establish backside engagement as a critical feature of ATG8 lipidation and illuminate the dynamic architecture and regulation of ATG3.

Signal transducer and activator of transcription 3 (Stat3) is a key molecular target in many cancers due to its role in tumor cell proliferation and survival. T40214, a G-quadruplex (G4) forming oligonucleotide, targets the Stat3 dimer and inhibits its DNA binding activity. In this study, we introduced N²-furfuryl and N²-cinnamyl deoxyguanosine modifications at the G-tetrad positions in T40214 to assess their structural and antitumor effects in prostate cancer cells. A single N²-furfuryl/cinnamyl modification preserved the stable parallel G4 conformation. Incorporating either of these modifications into the top G-quartet (TF15 and TC15) significantly enhanced thermal stability. Molecular dynamics simulation studies revealed that the aryl moieties were well accommodated at the 5'-ends without disrupting the interaction with Stat3. It is also evident that H-bonding and π-π stacking interactions induced due to the presence of the aryl moieties contributed to the improved thermal stabilities for TF15 and TC15, respectively. Gel mobility assays confirmed that all aryl-modified T40214 G4s form stable 5'-5' dimers, similar to native T40214. TF15 and TC15 derivatives exhibited potent antiproliferative activity (IC₅₀ = 0.37-0.39 μM) and effectively induced apoptosis while suppressing the Stat3-mediated gene expressions in DU145 cells. Overall, these findings demonstrate the potential of these aryl modifications in T40214 as a promising Stat3-targeting therapeutic approach for prostate cancers.

In the AlphaFold era, there is a significant momentum in predicting protein structures, functionality, and mutational hotspots from deep learning approaches. In this review, we highlight how structural information is only a starting point in understanding function and why a single structure for a given sequence rarely captures the true picture. We provide an overview of selected experimental and computational techniques that can be employed to delineate the conformational landscapes of proteins at different levels of resolution. An integrative approach has often led to the identification of considerable heterogeneity in the native ensemble, with high-resolution methods revealing proportionately larger complexity. Partial structure in the native ensemble appears to be the norm, which typically appears as excited or intermediate states in the folding conformational landscape. A more nuanced approach mapping the ensemble of states, their relative populations, associated time scale of interconversion, and their sensitivity to different physical perturbations is therefore necessary. Thus, "sequence-ensemble-function" paradigm is the way forward even for apparently well-folded proteins, with multiprobe experiments and physically grounded models providing an intimate and intuitive understanding of this connection.

In general, the easier and cheaper the expression and purification processes are, the more profitable the production of a recombinant protein of interest is, especially in the industrial world. Previously, we have developed the lectinic CRD_SAT tag that we demonstrated is efficient at cost-effectively purifying passenger proteins. It also has the advantage of being quite small and limiting steric hindrance upon release by protease cleavage. Here, we used protein sequence optimization to design highly thermostable versions of CRD_SAT and showed that the midpoint denaturation temperature could be increased from 55.8 to 92.2 °C. In fact, our variants (called CRD_VLs) possess the ability to support a heating step during the purification process, which represents an easy way to eliminate thermolabile proteins coming from the host cells when the recombinant proteins are produced in bacteria. To challenge our CRD_VLs, we fused them to a PET hydrolase exhibiting promising industrial activity at 70 °C and showed that CRD_VL-tagged enzymes remain active, even after heating, with a balancing effect due to the CRD_VL internal mutations. Surprisingly, whereas mutations in CRD_VLs were introduced far from the d-lactose binding site, we also showed that the affinity toward the disaccharide was clearly improved in the variants to reach a dissociation constant (K_d) of around 30 μM (the K_d of the CRD_SAT being measured at around 90 μM), paving the way for the use of our tag in applications such as lectin-based affinity enrichment or those requiring few, cheap, and simple purification steps.

The conformational properties of alanine dipeptide (1) have been widely investigated by molecular dynamics (MD) and quantum mechanics calculations. Nine idealized conformers have been identified (α_L, α_D, C₇^ax, β₂, C₅, α', C₇^eq, P_II, and α_R), distinguished by their preferred backbone torsion angles φ (C_car-N-C_α-C_car) and ψ (N-C_α-C_car-N). The relative energies of these conformers differ depending on the force fields or the level of theory used in the calculations. MA'AT analysis has been applied to give experiment-based probability distributions of φ and ψ in 1 in an aqueous solution for comparison to those obtained by aqueous MD. Using ¹³C- and ¹⁵N-labeled isotopomers of 1, 11 redundant NMR J-couplings that depend primarily on either φ or ψ were measured from 1D and 2D NMR spectra. Density functional theory calculations were conducted to obtain potential energy surface (PES) plots, and φ- and ψ-dependent J-couplings were calculated as a function of both angles. Parameterized J-coupling equations were used in conjunction with experimental J-values in MA'AT analysis to give a reproducible average unimodal model of φ (298.8° and 43.8°; mean and circular standard deviation) and a tentative bimodal model of ψ (mean values of 192° ± 32° and 318° ± 22° (mean ± STD); both states are approximately equally populated). The experimental J-couplings indicate highly favored trans configurations of both amide bonds in 1. These findings support an experiment-based conformational model of 1 in aqueous solution involving α_R ⇌ P_II exchange, which is qualitatively consistent with PES and MD data.

The plant protein dehydrin of the late embryogenesis abundant (LEA) family plays an important role in abiotic stress tolerance. Here, we investigated the structural changes in DHN1 (dehydrin) protein from Zea mays (167 aa) upon exposure to varying temperatures and various concentrations of sodium dodecyl sulfate and trifluoroethanol, using spectroscopic techniques such as CD, fluorescence, and FRET. For this purpose, three mutants DHN1 CW1 (Cys⁶²─Trp¹²²), DHN1 CW2 (Cys⁶²─Trp¹³²), and DHN1 W3 (Trp³) were generated by replacing amino acids at sites between the two K-segments, near the S-segment and at the N-terminal. CD data revealed that DHN1 and its mutants CW1 and CW2 show substantial disorder to order (increased alpha helical content) transition on increasing temperature from 25 to 90 °C in contrast to the DHN1 W3 mutant. A large blue shift in tryptophan emission maxima, accompanied by rising Trp fluorescence anisotropy, increase in helical content accompanied by a reduction in the random coil structure of DHN1 revealed a transition from a disordered to ordered conformation in the presence of ∼0.2 mM SDS. Furthermore, intramolecular FRET between the Trp and Cys conjugated dansyl probe indicated that the distance between Trp¹²² and Cys⁶² in DHN1 CW1 was reduced from 34 to 26 Å in the presence of ∼0.4 mM SDS, while the distance between Trp¹³² and Cys⁶² in DHN1 CW2 was reduced from 24 to 22 Å. The DHN1 mutants displayed reduced cryoprotection but robust heat protection activity, with altered sensitivity to heat/SDS. Our results yield quantitative insights on the role of the N-terminal and K-segment, facilitating the folding of DHN1 triggered by exposure to anionic monomers of SDS.