Protein Science最新文献

Acylpeptide hydrolase (APEH) or acylaminoacyl-peptidase (AAP) is a serine hydrolase that regulates protein metabolism. It can also bind to and process unusual substrates, acting as a detoxifier. To better understand its promiscuous specificity, we determined the cryo-EM structures of mammalian APEH complexed with classical serine protease partners: a chloromethyl-ketone (CMK) inhibitor, an organophosphate (OP) pesticide (dichlorvos), and benzenesulfonyl-fluoride. Since CMK derivatives of N-acetylated peptides were suggested to induce apoptosis by inhibiting APEH, while OP complexes may serve as biomarkers of OP exposure and are linked to cognitive enhancement, these complexes carry physiological significance. We identified a unique strand-breaker Pro residue in the hydrolase domain, which relaxes the active site into a partially inactivated but more spacious conformation, transforming the classical serine protease apparatus into a versatile yet potent hydrolysis center with broad specificity, distinguishing the mammalian enzyme not only from other APEHs but also from serine α/β hydrolases sharing essentially the same fold.

The present work proposes an explanation for a recent observation that has conclusively proven that heavy water, that is, water containing the non-radioactive isotope of hydrogen deuterium, is mildly sweet, at variance with the tasteless common water. No firm explanation was proposed for this unexpected behavior. Yet, the subject is far from being an irrelevant curiosity, as the explanation of yet unidentified properties of the sweet receptor can help us to understand the molecular bases of food appreciation that have direct repercussions on pathologies such as diabetes and obesity. Here, a simple but convincing structural explanation of the taste of heavy water is proposed that is based on the influence of heavy water on the conformation of the active form of the receptor. The explanation requires the concept of "constitutive receptor activity", that is, a notion well accepted in many areas of pharmacology but clearly neglected in reference to taste receptors. We discuss how constitutive activity also explains other properties such as the recognition of sweet proteins that are several thousand times sweeter than small carbohydrates.

Following the SARS-CoV-2 pandemic, many direct-acting antivirals targeting viral cysteine protease were developed. SARS-CoV-2, as well as other viruses, rely on cysteine proteases for their replication, suggesting future generations of antivirals targeting cysteine proteases will emerge. A major concern for these first-generation drugs is the off-target effects on host cysteine proteases. Therefore, screening for inhibitor specificity is a crucial step in antiviral drug development. Cathepsins are one of the most abundant human proteases, which have roles in maintaining cell health and are key to many physiological processes. Here we describe a general expression and purification protocol for cathepsins B, S, and L using the Expi293™ mammalian expression system. We characterized the glycosylation pattern and kinetic parameters of purified enzymes with commercially available cathepsin-specific fluorogenic substrates and also established cathepsin inhibition screen assays. We tested specificity indices of peptidic inhibitors of SARS-CoV-2 M^pro synthesized by our team and nirmatrelvir as a benchmark for all three cathepsins. Establishing a reliable cathepsin inhibition assay would assist with screening of newly developed cysteine protease inhibitors for off-target activity within the scope of pandemic preparedness.

ADAR1p150 is a critical RNA-editing enzyme for maintaining cellular homeostasis through dsRNA binding, protein-protein interactions, and adenosine-to-inosine (A-to-I) editing. Beyond its dsRNA binding domains, ADAR1p150 contains a Zα domain that can induce a conformational switch of dsDNA and dsRNA from stable B/A forms to a higher-energy left-handed Z form. By stabilizing Z-RNA, ADAR1p150 is thought to modulate immune activation by competing with dsRNA sensors like MDA5 and ZBP1. ADAR1p150's editing activity minimizes dsRNA's presence and prevents dsRNA-mediated inflammatory pathways' activation. Our study employs NMR to introduce a novel pharmacophore model for Zα domain binders. We identify cromoglicic acid, also known as the FDA-approved drug cromolyn, as an ADAR1 Zα binder that competes with nucleic acid recognition. Cromolyn does not bind to ZBP1 Zα domains, which are the only other human Zα domains. Our work paves the way for effectively modulating ADAR1p150 function with small molecules, opening new avenues for enhancing anti-cancer immune responses.

Water is essential for active life, yet some organisms, such as tardigrades, can survive prolonged periods of drying-induced dormancy. Cytoplasmic abundant heat-soluble (CAHS) proteins are disordered proteins that undergo a phase transition from the solution to gel state. CAHS proteins help tardigrades survive extreme drying, increase hyperosmotic stress tolerance in heterologous systems, and preserve the function of labile enzymes during drying in vitro. It has been speculated that the ability of CAHS proteins to form gels might be mechanistically linked to their protective capacity. However, recent evidence suggests that while gelation enhances hyperosmotic stress tolerance, it is not required for this phenomenon. Still, the extent to which gelation is necessary for other CAHS-based protective functions, such as enzyme protection during drying, is unknown. Here, we show that rather than the solution or gel state of CAHS proteins being the sole protective phase, each phase is optimized to protect different enzymes during drying. Using in vitro assays that provide clear functional readouts and allow for precise control over CAHS and client enzyme ratios, we show that the gelled state of CAHS D, a model CAHS protein, promotes the protection of the enzyme lactate dehydrogenase during drying. We find that the opposite is true for the enzyme citrate synthase, with variants of CAHS D that do not gel providing optimal protection to this enzyme. Correlative analysis between protective capacity and sequence/ensemble features of CAHS D variants supports the notion that phase is a major driver of differential enzyme protection. Finally, we show that enhanced water binding is an emergent property of gelation that positively correlates with the protein's ability to protect LDH. These results demonstrate a link between the phase of CAHS proteins and their protective function, providing insights into how CAHS proteins help tardigrades counteract the spectrum of stresses encountered during different stages of drying. Broadly, this study advances our understanding of desiccation tolerance, while providing insights into engineering strategies to tune protein-based excipients to protect specific clients. This study contributes to a broader discussion in the protein field about the functionality of phase behavior and states.

Polyethylene terephthalate (PET) is one of the most significant plastic pollutants. Unlike other plastic polymers, PET can be degraded by PET-hydrolytic enzymes (PETases). Over the past two decades, numerous publications have reported the discovery, characterization, and engineering of PETases. This review thoroughly examines the sequence, structure, and functional diversity of naturally occurring PETases. To achieve this, we compiled data from 48 publications into a single table. The resulting dataset enabled us to contextualize previously reported features and shed light on the sequence-structure-function relationships of PETases. Finally, we review selected engineering campaigns and suggest future directions for the enzymatic recycling of PET under mesophilic and thermophilic conditions, aiming to understand the gaps to tackle the PET pollution crisis.

Kinked- $\beta$ sheets are short peptide motifs that appear as distortions in $\beta$ strands and often mediate formation of reversible amyloid fibrils in prion-like proteins. Standard methods for assigning secondary structures cannot distinguish these esoteric motifs. Here, we provide a supervised machine learning-based structural quantification map to unambiguously characterize kinked- $\beta$ sheets from coordinate data. We find that these motifs, although deviating from standard $\beta$ strand region of the Ramachandran plot, scatter around the allowed regions. We also demonstrate the applicability of our technique in wresting out LARKS, which are kinked- $\beta$ strands with designated sequence. Additionally, from our exhaustive simulation generated conformations, we create a repository of potential kinked peptide-segments that can be used as a screening-library for assigning $\beta$ kinks in unresolved coordinate dataset. Overall, our map for kinked- $\beta$ provides a robust framework for detailed structural and kinetics investigation of these important motifs in prion-like proteins that lead to formation of amyloid fibrils.

Nonenveloped viruses package, carry, and deliver their genomes to the targeted cells using protein shells known as capsids. The viral capsids come in different shapes and sizes, most exhibiting helical or icosahedral symmetries. Here, we analyzed 634 icosahedral capsids at high resolution (<4 Å) from 39 virus families with T-numbers ranging from 1 to 9 and evaluated the aggregated buried surface areas (BSAs) at the unique interfaces as a measure of capsid strength and protein-protein interactions (PPIs). The BSAs were further analyzed relative to their capsid diameters and the calculated molecular weight (MW) of coat protein subunits (CPs) occupying the icosahedral asymmetric unit (IAU). Our results show that naturally occurring viral capsids exhibit stronger PPIs relative to non-native and/or engineered capsids. Interestingly, the "T = 2" capsids cluster distinctly, exhibiting weaker PPIs relative to their capsid size and subunit MWs. Furthermore, the normalized BSAs by the MW of the CPs present in the IAU are fairly constant across different capsids, suggesting that the extent of the PPIs is proportional to the CP size with a few exceptions (e.g., "T = 2" capsids). We also identified the range of capsid diameters and MWs of CPs forming different T = number capsids, which suggest a CP of 30-50 kDa can be used to build any quasi-equivalent capsid with T-numbers 1-9. Furthermore, we identified the strongest capsids available at various diameters at 25 Å intervals. Taken together, in addition to the targeting specificities, the results from this study are useful for choosing viral capsids for biomedical applications.

In the blood-brain barrier (BBB), endothelial cells are joined by tight junctions (TJs), multi-protein assemblies that seal the paracellular space and restrict molecular transport. Among the BBB TJ proteins, Claudin-5 (Cldn15) is the most abundant one. Structural models for claudin complexes, first introduced for channel-forming, selectively permeable claudins, comprise protomers arranged to form paracellular pores that regulate transport by electrostatic and/or steric effects arising from pore-lining residues. With limited exceptions, computational studies explored oligomers of only a few subunits, while TJs are formed by extended polymeric strands. Here, we employ multi-microsecond all-atom molecular dynamics and free-energy (FE) calculations to study two distinct models of TJ-forming Cldn15 complexes, called multi-Pore I and multi-Pore II, each comprising 16 protomers arranged around three adjacent pores. FE calculations of water and ions permeation reveal that, in both models, ion transport is hindered by FE barriers higher than in single pores. Moreover, only the multi-Pore I model captures the Cldn15 G60R variant's effect, making it anion-permeable. The results provide insights into Cldn15 structure and function and validate a structural model of BBB TJs useful for studying barrier impairment in brain diseases and for developing therapeutic approaches.

The pyrroloindole (hexahydropyrrolo[2,3-b]indole, HPI) structural motif is present in a wide range of natural products with various biological activities, yet its chemical synthesis poses a challenge, particularly regarding methylation at the indole C3 position. In nature, S-adenosyl methionine (SAM)-dependent methyltransferases efficiently catalyze this reaction with high stereoselectivity. This study presents the investigation and rational re-design of a potential methyltransferase, termed SeMT, from the actinomycete Saccharopolyspora erythraea. While its three-dimensional structure elucidated via X-ray crystallography confirmed extensive structural similarity to cyclic dipeptide-processing methyltransferases such as SgMT, its putative catalytic center is clearly divergent. Accordingly, wild-type SeMT displayed minimal activity with diketopiperazine (DKP) substrates, triggering an extensive mutagenesis effort aimed at iteratively enhancing this methyltransferase function. This work yielded a variant with appreciable activity, which was comprehensively characterized. Notably, a specific mutation within the catalytic triad of SeMT proved critical not only for its own function but also for the temperature-activity profile of its homolog protein SgMT. Beyond the specific properties of SeMT, these findings hence provide important insights into the active center architecture of indole C3-methyltransferases, supporting further development of these enzymes into refined biocatalysts for synthetic applications.