Protein Science最新文献

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of many authorized monoclonal antibody products. This highlights the need for alternative strategies, especially for vulnerable populations such as immunocompromised individuals. Here, we optimized angiotensin-converting enzyme 2 (ACE2)-Fc fusion proteins by combining three engineering steps: in silico mutagenesis of the S protein binding interface to increase affinity, insertion of a flexible linker to improve protein stability and S protein accessibility, and generation of a tetrameric molecule to maximize avidity. Neutralizing activity was tested against a large panel of pre-Omicron and Omicron pseudoviruses and authentic viruses, including JN.1 and KP.2 variants. Optimized ACE2-Fc molecules demonstrated potent neutralizing activity, in the picomolar range, against all SARS-CoV-2 variants. Our molecules displayed similar potency but better resilience when compared to the monoclonal antibody Sipavibart. These findings support ACE2-Fc proteins as robust candidates for next-generation interventions against infection by an evolving SARS-CoV-2.

The HIV-1 Vpu membrane protein is crucial to the virus lifecycle. Our recent studies revealed soluble Vpu oligomers, prompting further investigation into their interactions with cellular proteins. Notably, Vpu may form a complex with calmodulin (CaM) due to its putative CaM-binding motif; however, experimental proof of this association is unavailable. Here, we present definitive experimental evidence that the soluble Vpu complex interacts in vitro with calcium-bound CaM (Ca²⁺-CaM), its active form. Using double electron-electron resonance (DEER) spectroscopy and protein spin labeling, we detected the formation of a soluble Vpu-Ca²⁺-CaM complex. Both the full-length (FL) and truncated C-terminal regions of Vpu bind Ca²⁺-CaM. DEER experiments on a spin-labeled CaM cysteine mutant S39C/A103C revealed that, upon association with Vpu, Ca²⁺-CaM undergoes a transition from an open to a more closed conformation, consistent with previous reports of Ca²⁺-CaM interactions with other proteins. Furthermore, we observed that the binding of Vpu to Ca²⁺-CaM leads to dissociation of soluble Vpu oligomers, as evidenced by a reduction in DEER modulation depth for FL Vpu spin-labeled at residue L42C. FRET analysis with a fluorescently labeled C-terminal cysteine mutant of Vpu confirmed this result. Like FL Vpu, the Vpu C-terminal region forms soluble homooligomers that dissociate upon binding to Ca²⁺-CaM. Collectively, our results suggest that soluble Vpu and Ca²⁺-CaM form an equimolar complex. DEER analysis of the Vpu C-terminal region spin-labeled at residues Q36C/I61C demonstrated that Vpu undergoes significant conformational changes to facilitate Ca²⁺-CaM binding. These findings could be relevant to Vpu-CaM interactions under physiological conditions.

Avian eggshell membrane (ESM) is fabricated within the isthmus region of the oviduct and is comprised of three juxtaposed, predominantly proteinaceous layers lying between egg white and the calcified shell. The limiting membrane is less than 0.5 μm in thickness and forms the osmotic barrier for the egg. This first layer provides the foundation for the successive deposition of two mats of protein fibers. Fibers from both inner and outer layers appear to have similar amino acid compositions and are notably disulfide-rich (comprising about 10% Cys). ESM has been utilized in a wide variety of applications, including nutraceutical supplements, tissue engineering, and nanofabrication, and yet fundamental questions concerning protein composition, fiber structure, and membrane assembly remain to be resolved. We previously identified an abundant disulfide-rich structural protein in chicken ESM fibers (cysteine-rich eggshell membrane protein; CREMP) that contains multiple tandemly repeated modules. In this work, we determine a structural model for four consecutive 2-disulfide containing CREMP modules using a variety of two- and three-dimensional solution NMR experiments. CREMP modules feature an N-terminal loop region positioned above a small beta hairpin that is stabilized by a conserved pattern of disulfide bridges between Cys1-3 and Cys2-4. While the individual CREMP modules are highly ordered, the lack of long-range inter-module restraints suggests an extended structure connected by flexible linkers. Finally, the structural information obtained in this work is considered in the context of full-length CREMP proteins and compared to two other structural proteins that contain multiple tandem repeats of 2-disulfide modules.

Macromolecular crowding is ubiquitous in physiological environments, perturbing the thermodynamics and kinetics of proteins via excluded volume and nonspecific chemical interactions. While crowding has been well studied in vitro and in cells, the inert sugar polymers used to simulate crowding lack the chemical characteristics of biomolecules. Emerging studies guide the development of more relevant models of crowding in the cell, but little work has been done to discern crowding effects on proteins at the cell surface. Using ¹⁹F NMR, we measure how protein stability, folding, and intermolecular interactions are modulated by three glycopolymers abundant at the cellular exterior. Biologically relevant glycopolymers including heparin, hyaluronic acid, and mucin significantly stabilize the folding of the N-terminal domain of the Drk-SH3 protein. These interactions are enthalpically stabilizing, emphasizing the importance of chemical interactions for biologically relevant crowders. We further show that these glycopolymers stabilize a homodimer formed by the A34F variant of GB1, demonstrating that biological crowders not only affect isolated proteins, but also influence how proteins interact with one another. Crowding is more complex than simple ideas of volume exclusion suggest, and our work guides a more comprehensive understanding of protein crowding in the context of the glycocalyx, the last frontier of the cell.

Liquid-liquid phase separation (LLPS) is emerging as a key mechanism for organizing cellular components and regulating stress responses. Although LLPS has been extensively studied in intrinsically disordered proteins, whether highly charged and intrinsically disordered molecular chaperones undergo LLPS remains poorly understood. Here, we demonstrate that the Escherichia coli acid shock protein Asr, a highly charged and intrinsically disordered chaperone, undergoes LLPS driven by electrostatic interactions and forms dynamic liquid condensates with polyanions such as DNA, RNA, heparin, and acidic proteins. Asr phase separation critically depends on positively charged clusters, polyanion length, ionic strength, and pH. Guided by Asr's physicochemical features, we identify three additional molecular chaperones, Anhydrin, Hero7, and HCVncd, that also exhibit LLPS behavior in vitro but display distinct condensate properties and pH responsiveness consistent with their individual charge compositions and distributions. In vivo, Asr-EGFP forms non-canonical compartments in 37% of E. coli cells at pH 7.5, increasing to 80% under acidic conditions (pH 4.5). These compartments disassemble under high-salt conditions after cell lysis, suggesting electrostatic mediation. In cell imaging and FRAP analyses further reveal that charge-enhanced Asr mutants and homologs form canonical condensates in vivo, predominantly co-localizing with acidic proteins. Notably, Asr*3 fusion drives condensate formation of the aggregation-prone client thereby reducing stress-induced aggregation, indicating that Asr functions as an LLPS-promoting module to mitigate protein aggregation. These findings advance our understanding of LLPS in highly charged, intrinsically disordered molecular chaperones and lay the foundation for exploring their roles in cellular homeostasis and potential applications in engineering synthetic biomolecular condensates.

High-temperature requirement A (HtrA) proteases are a conserved family of serine proteases central to protein quality control and bacterial virulence. While Gram-negative and human HtrAs are structurally well studied, Gram-positive homologs remain essentially uncharacterized. Here, we present the first integrated structural and mechanistic analysis of a Gram-positive HtrA, from Streptococcus pneumoniae, a virulence factor essential for adhesion and infection in vivo. Proteomic profiling of an htrA knockout and cleavage assays demonstrate that S. pneumoniae HtrA is required for protein quality control, with the PDZ domain mediating substrate recognition. Biochemically, S. pneumoniae HtrA exists exclusively as a monomer in solution, a striking divergence from canonical trimeric HtrAs that we show is shared with other Gram-positive homologs. NMR analyses reveal that the monomer dynamically samples open and closed conformations, while cryo-EM of a catalytic mutant identifies a hexamer stabilized by a unique LoopA-PDZ interaction. Together, these findings define S. pneumoniae HtrA as a dynamic monomer with interdomain coupling between its protease and PDZ domains, establishing Gram-positive HtrAs as a mechanistically divergent subgroup within the HtrA family.

Curli, which are the major proteinaceous components of the Escherichia coli biofilm extracellular matrix, help protect cells against environmental stressors, including dehydration and antibiotics. Composed of the amyloid proteins CsgA and CsgB, curli self-assemble as these protomers are secreted into the extracellular space. The mechanisms of curli assembly and their functional roles within the extracellular matrix are incompletely understood. High-resolution imaging tools compatible with live-cell conditions provide a critical means to investigate the assembly and function of curli in their native context. In this study, we use super-resolution imaging to visualize curli fibrils on living bacterial cells. Transient amyloid binding of the fluorogenic dye Nile blue facilitates two complementary super-resolution fluorescence microscopy approaches: single-molecule imaging via points accumulation for imaging in nanoscale topography and super-resolution optical fluctuation imaging via pixel-wise autocorrelation. Additionally, imaging fluorescence correlation spectroscopy was used to measure the characteristic relaxation times associated with Nile blue binding to CsgA fibrils. Together, these approaches offer a framework for imaging-based biophysical characterization of curli structures on living cells.

OX40 and OX40L belong to the tumor necrosis factor receptor superfamily (TNFRSF) and tumor necrosis factor superfamily (TNFSF), respectively. Protein-protein interactions between OX40 and OX40L facilitate T cell responses, triggering various immunological and pathophysiological events. Excessive activation frequently contributes to the onset of autoimmune and allergic diseases. Therefore, the OX40/OX40L system is considered a promising target for drug discovery. Given that the structure of the OX40-OX40L complex exhibits some unique features compared to other members of these protein super families, it is reasonable to assume that this tandem possesses distinct interaction mechanisms. However, detailed interaction analysis using quantitative parameters such as binding kinetics or thermodynamics, with remains to be performed for OX40/OX40L. In this study, we identified several hot spot residues from the OX40 cysteine-rich domains (CRDs) 1 to 3 by alanine scanning. Kinetic and thermodynamic analysis combined with molecular dynamics simulations highlighted the characteristics of a hot spot from CRD3 due to its indirect influence on those from CRD1 and CRD2, providing insights into the interaction mechanism and a strategy for drug discovery targeting this interaction.

Eukaryotic elongation factor 2 kinase (eEF-2K) is a member of the α-kinase family of atypical serine/threonine kinases. eEF-2K, the only calmodulin-activated α-kinase, phosphorylates the ribosome-associated GTPase, eukaryotic elongation factor 2 (eEF-2), suppressing translational elongation. α-kinases, including eEF-2K, possess catalytic site geometries that are distinct from those of typical kinases, suggesting possible divergence in their phospho-transfer mechanisms. Unlike typical protein kinases, where chemistry is known to proceed through a sequential mechanism involving a ternary kinase-substrate-ATP•Mg²⁺ complex, the nature of the chemical step catalyzed by α-kinases remains poorly defined. Here, multiple orthogonal lines of evidence, including a crystal structure and solution-state mass spectrometry data, suggest phosphorylation of a catalytically essential aspartate residue (D284) at the eEF-2K active site. Previous crystallographic evidence of the presence of a phospho-aspartate at an equivalent position (D766) in the related Dictyostelium α-kinase MHCK-A strongly suggests that this species represents a conserved active-site feature in α-kinase family members, despite their disparate modes of activation. This observation, together with existing kinetics data on eEF-2K, raises the possibility that phospho-transfer chemistry in α-kinases occurs via an ordered stepwise mechanism involving a phospho-enzyme intermediate, contrasting with typical protein kinases.

Here, we investigate the effects of glycosylation at position N99 on the structural dynamics and lipid scrambling activity of ATG9A, a key autophagy protein, using microsecond all-atom molecular dynamics simulations. ATG9A is an integral membrane protein involved in autophagosome biogenesis, and glycosylation at N99 has previously been implicated in intracellular trafficking, although its precise role remains unclear. The simulations reveal that the hydrophilic central cavity of ATG9A supports lipid reorientation and partial trans-bilayer movements, consistent with experiments on its lipid scrambling activity. We propose that N-glycosylation at N99 enhances cooperative interactions between protomers, facilitating lipid insertion and translocation within the central cavity. These findings suggest a mechanism by which glycosylation may influence lipid redistribution across the phagophore membrane during autophagy. To test this hypothesis, we generate N99 variants (ATG9A^N99A and ATG9A^N99D) lacking N-glycosylation. These mutants show no significant changes in autophagy flux, suggesting that N99 glycosylation may not be essential for bulk autophagic processing. However, the analysis of autophagosome size indicates that the variants fail to rescue the enlarged vesicle phenotype of ATG9A-KO cells, unlike wild-type ATG9A. Thus, glycosylation might fine-tune ATG9A function, influencing vesicle morphology through conformational dynamics and lipid transport. We also observe asymmetric protomer conformations in ATG9A, in contrast to the symmetric structures obtained from cryo-EM, suggesting that structural heterogeneity could be further explored with experimental methods. Overall, our study highlights the importance of including glycosylation in computational models of membrane proteins and provides mechanistic insight into lipid transport during autophagy, with potential implications for other lipid scramblases and flippases.