Protein Science最新文献

Many peptide drugs rely on nonproteinogenic amino acids and chemical modifications for improved activity and proteolytic stability. However, these features also make drug production expensive and challenging to scale. Here, we engineered small, linear, proteinogenic peptides that bind human programmed death-ligand 1 (hPD-L1) with high affinity and stability using mRNA display affinity maturation. The resulting peptides, SPAM2 and SPAM3, have antibody-like affinities for hPD-L1 (dissociation constants between ~250 and 300 pM) and are selective for hPD-L1. Both SPAM2 and SPAM3 compete with hPD-L1 ligands known to interact with the programmed cell death protein 1 site and are stable in human serum. SPAM3 bound human glioma D423 cells with high affinity in flow cytometry experiments comparable to that of a clinical therapeutic antibody. These results support the use of affinity maturation selections to dramatically enhance the biophysical properties of linear, proteinogenic peptides for translational applications.

Hydrophobins are a family of small fungal proteins that self-assemble at hydrophobic-hydrophilic interfaces. Hydrophobins not only play crucial roles in filamentous fungal growth and development but also have attracted substantial attention due to their unique material properties. Structural characterization of class I and class II hydrophobins to date has been limited to a handful of proteins. While machine-learning-based structure prediction methods have the potential to exponentially expand our ability to define global structure-function relationships of biomolecules, they have not yet been extensively applied to hydrophobins. Here, we apply a suite of bioinformatics tools including Rosetta, AlphaFold, FoldMason, and Foldseek toward analysis, modeling, classification, and global comparison of class I and class II hydrophobins. We first probe the structural and energetic features of experimental class I and class II structures available in the Protein Data Bank. Using previously solved X-ray and NMR structures, we benchmark the ability of AlphaFold to predict class I and class II hydrophobin folds. We explore the physicochemical properties of more than 7,000 class I and class II hydrophobins in the UniProt database. Then, using AlphaFold models, we classify the structural universe of all known class I and class II hydrophobins into six distinct clades. We also uncover putative non-canonical features of hydrophobins, including extended N-terminal tails, five disulfide bonds, polyhydrophobins, and non-hydrophobin proteins containing hydrophobin-like folds. Finally, we examine the ability of AlphaFold and Chai-1 to model hydrophobin membrane binding, conformational changes, and self-assembly of class I rodlets and class II meshes. Together, our results highlight that AlphaFold not only accurately models and enables the global comparison of features within the hydrophobin protein family but also uncovers new properties that can be further evaluated with experimentation.

Zinc finger (ZF) proteins are the most abundant transcription factors in vertebrates, and they regulate gene expression through interactions with cis-acting elements. ZF domains selectively recognize specific sequences to accelerate or repress target genes. Zinc finger protein 18 (ZNF18) contains five CX₂CX₁₂HX₃H-type ZFs at the C-terminus, which are expressed in the brain and other organs of the biological system. Bioinformatic study proposed that cyclin-dependent kinase 1 (CDK1) is in the signaling cascade of ZNF18; although experimental evidence has not yet been reported. In this study, we expressed and purified ZNF18(ZF1-5), five ZF domains from ZNF18, and investigated metal binding specificity and promoter interactions. ZNF18(ZF1-5) has specific coordination to Zn²⁺ (K_d ≤ 18 nM) compared with other xenobiotic metal ions, including Co²⁺, Fe²⁺, and Fe³⁺, with 98.5% of reduced ZF domains after purification. This significantly active ZF can be one of the major reasons for tight coordination affinity. CDK1 rescued the arrested cell cycle induced by DNA damage, resulting in tumorigenesis. Zn²⁺-ZNF18(ZF1-5) specifically binds to cis-acting elements of cdk1 (K_d = 4.63 ± 0.07 nM), mediated by a cell cycle-dependent element (cde, 5'-CGCGG) and a cell cycle gene homology region (chr, 5'-TTGAA). The ZNF18 superfamily was expressed in the brain for the regulation of neuronal development and cell differentiation. Zn²⁺-ZNF18(ZF1-5) interacted with promoters in the insulin response sequence (IRS) for inhibition of dopamine secretion and cis-acting element of brain-2 (BRN2), which controlled astrocyte and cancer development. These results provide the first evidence that ZNF18(ZF1-5) regulates the cell cycle and neuronal development through transcriptional regulation.

β-Glucosidases, major enzymes that release glucose from various natural compounds, are phylogenetically classified into glycoside hydrolase (GH) families. GH1 is the largest of these families. No β-1,2-glucan-associated GH1 enzyme has been found, even though β-1,2-glucans are natural carbohydrates that are important for interaction between organisms and environmental adaptation. In this study, functional and structural analyses of a GH1 enzyme from Streptomyces griseus (SGR_2426 protein) were performed. SGR_2426 showed the highest hydrolytic activity toward p-nitrophenyl β-glucopyranoside among p-nitrophenyl sugars. This enzyme showed hydrolytic activity toward β-1,2-glucooligosaccharides specifically among β-linked glucooligosaccharides. A structure of the enzyme in complex with sophorose (β-1,2-glucodisaccharide) was obtained as a Michaelis complex. The six-membered ring of the glucose unit at the reducing end of sophorose is positioned in a hydrophobic environment between Trp291 and Met171, while only residue Gln229 forms a hydrogen bond directly. Trp291 and Gln229 are proposed as candidates for the residues important for substrate specificity based on comparison with structurally characterized GH1 homologs. Mutational analysis of Trp291 and Gln229 suggested that Trp291 is important for substrate recognition but not for substrate specificity and that Gln229 is involved in substrate specificity. SGR_2426 is the first identified β-1,2-glucan-associated β-glucosidase in the GH1 family.

Structures based on x-ray diffraction data collected at 2.3, 2.88, and 2.95 Å resolutions have been determined for long-acting dihexamer insulin at three different temperatures, ranging from 100 to 300 K. It has been observed that the unit-cell parameters of the insulin crystal at 100 K change at 200 K. This change is likely due to the subtle repacking of the rhombohedral insulin crystal and the loss of noncovalent interactions involving myristic acid, which binds two hexamers. Computational analyses indicate that allosteric residues and fatty acid-binding residues of insulin hexamers exhibit reduced collective dynamics and inter-residue coupling, possibly resulting from increased structural fluctuations due to elevated thermal vibrations. This transition has been observed at a characteristic temperature of 200 K, potentially highlighting underlying alterations in the dynamic structure of the fatty acid-solvent interface in the dimer of hexamers. Combined with computational analyses, these findings provide key insights into thermal stability mechanisms, which are crucial for developing thermostable insulin formulations in industrial applications.

Metabotropic glutamate (mGlu) receptors play a crucial role in synaptic transmission through homodimeric or heterodimeric assemblies. Despite their dimeric nature, only one subunit within the mGlu dimer engages with G proteins during activation, and the biased activation can be further controlled by allosteric modulators. Considering the related molecular mechanisms remain elusive, we employed Gaussian accelerated molecular dynamics (GaMD) simulations to investigate the regulated mechanisms in mGlu₂-mGlu₄ heterodimers. Our results demonstrate that the G_i protein exhibits a higher binding affinity for mGlu₄ compared to mGlu₂ within the mGlu₂-mGlu₄ heterodimer. Meanwhile, when the positive allosteric modulator (PAM) binds to G_i-coupled subunits-whether mGlu₂ or mGlu₄-it can enhance the binding affinity between the G_i protein and the subunits of the mGlu₂-mGlu₄ heterodimer. However, if the PAM binds to mGlu₂ while the G_i protein is coupled to mGlu₄, the binding affinity may be reduced. Additionally, our results highlight the crucial role of the ICL2 region and the perturbation of the residue-residue coupling network involved in the regulatory pathways in mediating the PAM-induced modulation of G_i protein preference. In conclusion, these findings provide novel insights into the molecular mechanism underpinning the G_i protein's preference for mGlu₄ within the mGlu₂-mGlu₄ heterodimers and the regulatory influence of PAM on G_i protein binding, advancing our understanding of their functional mechanisms.

The emergence of SARS-CoV-2 and other lethal coronaviruses has prompted extensive research into targeted antiviral treatments, particularly focusing on the viral 3C-like protease (3CL^pro) due to its essential role for viral replication. However, the rise of drug resistance mutations poses threats to public health and underscores the need to predict resistance mutations and understand the mechanism of how these mutations confer resistance. The binding of inhibitor to 3CL^pro drives it from the monomeric to the active dimeric form, which can counterintuitively lead to enzyme activation rather than inhibition. Furthermore, we find this allosteric coupling between binding and dimerization is sensitive to mutation, leading to a new mechanism for drug resistance. Understanding the relationship between inhibitor binding and dimerization is important for resistant strain surveillance and development of robust antivirals. Herein, we present a systematic study of drug resistance mediated by inhibitor-induced dimerization of 3CL^pro.

Adherens junctions (AJs) are essential for maintaining tissue integrity and regulating intercellular signaling and tumor progression. At the core of AJs is the cadherin-catenin (ABE) complex, which links to the cytoskeletal actin filament (F-actin). Vinculin, a cytoskeletal protein, is recruited to AJs under recurrently high tensions to modulate cell adhesion. However, the molecular mechanisms underlying vinculin recruitment and activation remain elusive due to the highly dynamic and heterogeneous nature of the cadherin-catenin-vinculin (VABE) complex. In this study, we performed molecular dynamics (MD) simulations to probe the structure, dynamics, and domain interactions within the VABE complex. Our simulations reveal that vinculin binding enhances the conformational flexibility of α-catenin and expands the configurational space sampled by its actin-binding domain (ABD). This is consistent with an increase in configurational entropy upon complex assembly, suggesting that an entropic trap mechanism-previously proposed for ABE-may also underlie force-sensitive binding in the VABE complex. Furthermore, we provide detailed structural insights into α-catenin/vinculin and α-catenin/β-catenin interactions, elucidating how vinculin recruitment impacts the dynamics of the ABE complex. Interestingly, while vinculin binding increases overall structural fluctuations, ABD exposure remains comparable to that of the ABE complex alone. This is likely due to ABD's interaction with the M1 subdomain, which emerges from α-catenin unfolding upon vinculin binding. Together, these findings deepen our understanding of vinculin-mediated mechanotransduction at AJs and its role in modulating cytoskeletal dynamics.

Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in many solid tumors, which is known to interact with cancer antigen CA125/MUC16, promoting cancer cell adhesion and metastasis. MSLN has been used as a target of multiple antibody-based therapeutic strategies, but their efficacy remains limited, potentially due to inherent pharmacokinetics conferred by the structure of antibodies (~150 kDa). To provide an alternative targeting molecule, we engineered a small scaffold protein derived from the tenth domain of human fibronectin type III (Fn3, 12.8 kDa) to bind MSLN with nanomolar affinity as a theranostic agent for MSLN-positive cancers. In this study, we explored the Fn3-MSLN interaction site through computational modeling and experimentally validated the model through domain-level and fine epitope mapping. Fn3-MSLN binding was predicted by a consensus approach, comparing multiple protein-protein docking software, the deep-learning-based algorithm AlphaFold3, and performing molecular dynamics (MD) simulations. To validate the prediction, full-length MSLN, single MSLN domains, or combinations of domains were expressed on the yeast surface, and Fn3 binding to displayed MSLN domains was measured by flow cytometry. The employed algorithms predicted two distinct binding modes for Fn3. Overall, experimental data agreed with our in silico prediction resulting from the AlphaFold3 model, confirming that MSLN domains B and C are predominantly involved in the interaction.

Recent evidence suggests that peptide-RNA coacervates may have buffered the emergence of folded domains from flexible peptides. As primitive peptides were likely composed of both L- and D-amino acids, we hypothesized that coacervates may have also supported the emergence of chiral control. To test this hypothesis, we compared the coacervation propensities of an isotactic (homochiral) peptide and a syndiotactic (alternating chirality) peptide, both with an identical sequence derived from the ancient helix-hairpin-helix (HhH) motif. Using electron paramagnetic resonance (EPR) spectroscopy and atomistic molecular dynamics (MD) simulations, we found that the syndiotactic peptide does not form stable dimers with high α-helicity in solution, unlike the isotactic peptide. However, both peptides do coacervate with RNA, albeit with distinct reentrant phase behaviors. Coacervation in each case is facilitated by oligomer formation, likely dimerization, upon RNA binding that promotes RNA cross-linking. Additionally, RNA cross-linking and coacervation of the syndiotactic peptide may involve α-helical conformations, according to atomistic MD simulations. Coarse-grained MD simulations indicate that the differences in reentrant phase behavior of isotactic and syndiotactic peptides are associated with differences in dimer flexibility and stability, which modulate the strength of peptide-peptide and peptide-RNA interactions and, consequently, the effectiveness of RNA cross-linking. These results illustrate how RNA binding and/or coacervation by early proteins could have promoted the transition of flexible, heterochiral peptides into folded, homochiral domains.