Cytoplasmic incompatibility (CI) is induced by the toxin protein CifB from Wolbachia and related bacteria through its deubiquitinase (DUB) domain; however, the substrate recognition and catalytic mechanisms of this domain remain unclear. Here, we report the high-resolution crystal structure of the type V CifB DUB domain from Rickettsia felis (a close relative of Wolbachia) in complex with ubiquitin. We found that DUBR.felis adopts a canonical CE clan/Ulp1-like fold. Mutational analysis revealed that the catalytic residue Cys1152 is essential for enzymatic activity, whereas mutations of His1095 or Asp1114 only partially reduce activity. Structural analysis further suggests the presence of a water-mediated compensatory hydrogen-bonding network within the active site. Further analysis of the complex structure shows that the interaction between DUBR.felis and ubiquitin can be divided into three distinct interfaces. DUBR.felis undergoes conformational rearrangements via an induced-fit mechanism: the VR3 region moves outward to form a channel for the ubiquitin C-terminal Gly-Gly motif, while the VR2 loop undergoes an approximately 60° flip to remodel the substrate-binding pocket. These changes, together with a hydrogen-bonding network between catalytic His1095 and ubiquitin Gly76, enable precise substrate positioning and efficient cleavage. This study provides a theoretical framework for understanding the catalytic mechanism of type V CI factor DUBs and their role in CI.

Abstract Most known anti-CRISPR proteins exert inhibition via solitary functional pathways, and few natural Acr homologs have been reported to possess coupled inhibitory mechanisms. Here, we demonstrate that AcrIIC1 Boe represses Nme1Cas9 activity through loop-coupled inhibitory effects. Beyond canonical occlusion of the HNH catalytic site, the unique extended loop of AcrIIC1 Boe further perturbs sgRNA binding and restricts R-loop maturation to reinforce inhibition. Strikingly, loop deletion completely abrogated this layered inhibitory capacity, confirming that the loop is indispensable for full anti-CRISPR function. Consistently, transplantation of this functional loop into the weakly active AcrIIC1 Nme1 markedly upgraded its inhibitory performance. Our findings reveal an unprecedented loop-governed coupled inhibition mode, clarify the structural basis for functional divergence within the AcrIIC1 family, and provide a modular engineering strategy for the rational design and optimization of high-potency anti-CRISPR proteins.

Insect incompatibility technology and population replacement strategies based on the principle of cytoplasmic incompatibility (CI) have been proven to be effective in controlling mosquito-borne diseases. However, the genetic diversity of Wolbachia and the species-specificity of mosquito vectors lead to a range of CI phenotypes in the infected hosts. As key proteins regulating the CI phenomenon, CI factors (including CidA and CidB) play a critical role in this process. Studies have shown that the diversity of CI factors is closely related to the diversity of CI phenotypes, although the molecular mechanisms remain unclear. In this study, we systematically analyzed the interactions between 21 CidA types (wPip (I-IV)) and 4 CidB types (wPip-I), and identified key interaction sites located in two distinct regions of their interaction interfaces. Each region is composed of two distinct amino acid motifs. This finding suggests that different types of CidA and CidB can form four different families, resulting in 16 distinct interaction patterns. By predicting the structure of representative CidA-CidB complexes, we further analyzed the specific amino acid interaction residues and confirmed that this interaction pattern is conserved between CidA (wPip (I-IV)) and CidB (wPip (I-III)). Our findings provide an important theoretical basis and technical platform for the artificial manipulation and regulation of Wolbachia-induced CI. This work offers a new direction for the precise cross-species control of CI factors through sequence- and structure-guided molecular modification and design.

Extracellular ATP (eATP) has emerged as a crucial signaling molecule across eukaryotic and prokaryotic domains, modulating diverse cellular functions by activating purinergic receptors to initiate intracellular signaling cascades. However, the structural and molecular mechanisms underlying eATP sensing and signaling by prokaryotic receptors remain largely unknown. Here, we demonstrate that the receptor PA2072 in Pseudomonas aeruginosa is responsible for recognizing eATP to down-regulate intracellular cyclic di-GMP levels. The periplasmic CHASE4 domain of PA2072 specifically binds and hydrolyzes eATP, exhibiting ATPase activity both in the presence and absence of a divalent cation cofactor. Structural elucidation of the PA2072 CHASE4 domain in its monomeric and complex states unveils an exquisite molecular switch governed by the oligomeric state. ATP hydrolysis by the catalytically active monomeric form is coupled to homodimerization, concomitantly deactivating its ATPase activity and initiating intracellular phosphodiesterase activity. These findings open avenues for understanding interkingdom eATP signaling and developing targeted therapeutic interventions.

Mechanical regulation of neural stem cell behavior is crucial for cellular transplantation and neural regenerative medicine. However, how neural stem cells perceive and respond to mechanical signals remains to be fully understood. In this study, a GHz bulk acoustic wave (BAW) resonator-based acoustic streaming (AS) regulatory system was designed, aiming to generate tunable shear forces on the cells for the controlled regulation of neuroectodermal (NE-4C) stem cell behavior. Results demonstrated that the gradient shear force produced by AS exhibited controlled regulation of cell movement, which could promote the transformation of the movement mode of cells from pseudopodia into bleb-driven movement rapidly. Then, AS was found to enhance cell motility by approximately 9.8 times compared to the unstimulated group. It was further proved that short-term AS stimulation could stably and efficiently promote both the parallel and vertical migration of cells. The number of vertically migrated cells in the 20 min AS-stimulated group was 10.9 times higher than that of the unstimulated group. Finally, the data showed that the proliferation multiple of cells could be controlled by changing the AS stimulation time and the input power of the device. In addition, AS stimulation could significantly accelerate the formation of neurite processes, ultimately leading to the production of neurons. To sum up, the AS shear force regulation system opened up the possibility of channel-less microfluidic systems, which could easily manipulate the cellular morphological changes. It provided a flexible tool for controllably regulating the migration, proliferation, and differentiation of neural stem cells, demonstrating its great potential in the fields of neural tissue engineering and regenerative medicine.

The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. AFM-1 is a MBL derived from Alcaligenes faecalis and has 86% homology with the NDM-1 family. Both AFM-1 and NDM-1 demonstrate the capacity to hydrolyze ampicillin and other β-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key amino acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket of AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues in the Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised of hydrophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a novel theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs.

The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. 1 is a MBL derived from Alcaligenes faecalis and shares 86% homology with the NDM-1 family. Both AFM-1 NDM-1 demonstrate the ability to hydrolyze ampicillin and other (3-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised drophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs.

Wild -type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2 -fold higher than that of wild -type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 degrees C for 5 days. The residual enzymatic activity was 65fold higher than that of the wild -type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and 7C -7C stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester. It poses serious threat to terrestrial, aquatic ecosystems and human health since it is difficult to be broken down and deposited in the environment. The biodegradation based on enzymatic catalysis offers a sustainable method for recycling PET. A number of PET hydrolases have been discovered in the last 20 years, and protein engineering has increased their degradation capabilities. However, no PET hydrolases that are practical for widespread industrial use have been identified. Screening of PET hydrolase using conventional detection techniques is laborious and inefficient process. Effective detection techniques are required to promote the commercialization of PET hydrolases. Using efficient detection techniques to screen potent industrial enzymes is essential for supporting the widespread industrial implementation of PET hydrolases. To define PET hydrolase, scientists have created a number of analytical techniques recently. The detection techniques that can be used to screen PET hydrolase, including high performance liquid chromatography, ultraviolet absorption spectrometric, and fluorescence activated droplet sorting method, are summarized in this study along with their potential applications.

The process of recycling poly(ethylene terephthalate) (PET) remains a major challenge due to the enzymatic degradation of high-crystallinity PET (hcPET). Recently, a bacterial PET-degrading enzyme, PETase, was found to have the ability to degrade the hcPET, but with low enzymatic activity. Here we present an engineered whole-cell biocatalyst to simulate both the adsorption and degradation steps in the enzymatic degradation process of PETase to achieve the efficient degradation of hcPET. Our data shows that the adhesive unit hydrophobin and degradation unit PETase are functionally displayed on the surface of yeast cells. The turnover rate of the whole-cell biocatalyst toward hcPET (crystallinity of 45%) dramatically increases approximately 328.8-fold compared with that of purified PETase at 30 °C. In addition, molecular dynamics simulations explain how the enhanced adhesion can promote the enzymatic degradation of PET. This study demonstrates engineering the whole-cell catalyst is an efficient strategy for biodegradation of PET.

Cytoplasmic incompatibility (CI) results when Wolbachia bacteria-infected male insects mate with uninfected females, leading to embryonic lethality. “Rescue” of viability occurs if the female harbors the same Wolbachia strain. CI is caused by linked pairs of Wolbachia genes called CI factors (CifA and CifB). The co-evolution of CifA-CifB pairs may account in part for the incompatibility patterns documented in insects infected with different Wolbachia strains, but the molecular mechanisms remain elusive. Here, we use X-ray crystallography and AlphaFold to analyze the CI factors from Wolbachia strain w Mel called CidA w Mel and CidB w Mel . Substituting CidA w Mel interface residues with those from CidA w Pip (from strain w Pip) enables the mutant protein to bind CidB w Pip and rescue CidB w Pip -induced yeast growth defects, supporting the importance of CifA-CifB interaction in CI rescue. Sequence divergence in CidA w Pip and CidB w Pip proteins affects their pairwise interactions, which may help explain the complex incompatibility patterns of mosquitoes infected with different w Pip strains.

The existing zoonotic coronaviruses (CoVs) and viral genetic variants are important microbiological pathogens that cause severe disease in humans and animals. Currently, no effective broad-spectrum antiviral drugs against existing and emerging CoVs are available. The CoV main protease (Mpro) plays an essential role in viral replication, making it an ideal target for drug development. However, the structure of the Deltacoronavirus Mpro is still unavailable. Porcine deltacoronavirus (PDCoV) is a novel CoV that belongs to the genus Deltacoronavirus and causes atrophic enteritis, severe diarrhea, vomiting and dehydration in pigs. Here, we determined the structure of PDCoV Mpro complexed with a Michael acceptor inhibitor. Structural comparison showed that the backbone of PDCoV Mpro is similar to those of alpha-, beta- and gamma-CoV Mpros. The substrate-binding pocket of Mpro is well conserved in the subfamily Coronavirinae. In addition, we also observed that Mpros from the same genus adopted a similar conformation. Furthermore, the structure of PDCoV Mpro in complex with a Michael acceptor inhibitor revealed the mechanism of its inhibition of PDCoV Mpro. Our results provide a basis for the development of broad-spectrum antivirals against PDCoV and other CoVs.

在过去的10年中,以新德里金属β-内酰胺酶-1(NDM-1)为代表的金属β-内酰胺酶在全球范围内广泛传播,对公共卫生安全产生了较大的威胁.尤其是近些年这些酶的突变体的出现使得耐药菌给人类健康造成了更加复杂和困难的挑战.目前,临床上仍然缺乏有效的治疗药物和手段.研发有效广谱的抑制剂成为解决此问题的重点.因此本文将针对NDM-1及其相关抑制剂复合物的三维结构解析工作进行综述,希望从生物学机制研究的角度带给相关研究人员一点启发和帮助.

The important role of BV in clinical diagnostics of liver-related diseases has been established in veterinary medicine. However, the sensitivity and selectivity of the current BV assays remain relatively low compromising its wider application in clinical diagnosis. Herein, we developed a rapid and sensitive BV-detecting biosensor based on a novel far-red fluorescent protein smURFP, which produced fluorescence only through specific interaction with its cofactor BV. In our study, the binding of BV to smURFP was then systematically optimized based on the structures of the smURFP + BV complex to increase the sensitivity of our biosensor. A wide linear range from 0 μM to 25 μM was obtained in both chicken and human serum. The limit of detection (LOD) and limit of quantification (LOQ) for BV was as low as 0.4 nM and 1.5 nM in human serum, and 0.4 nM and 1.2 nM in chicken serum. To our knowledge, this is the lowest LOD that has ever been reported for a BV biosensor. Our study sheds light on the biological and clinical analysis of BV.

The flavivirus nonstructural protein 3 helicase (NS3hel) is a multifunctional domain protein that is associated with DNA/RNA helicase, nucleoside triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase) activities. As an NTPase-dependent superfamily 2 (SF2) member, NS3hel employs an NTP-driven motor force to unwind double-stranded RNA while translocating along single-stranded RNA and is extensively involved in the viral replication process. Although the structures of SF2 helicases are widely investigated as promising drug targets, the mechanism of energy transduction between NTP hydrolysis and the RNA binding sites in ZIKV NS3hel remains elusive. Here, we report the crystal structure of ZIKV NS3hel in complex with its natural substrates ATP-Mn2+ and ssRNA. Distinct from other members of the Flavivirus genus, ssRNA binding to ZIKV NS3hel induces relocation of the active water molecules and ATP-associated metal ions in the NTP hydrolysis active site, which promotes the hydrolysis of ATP and the production of AMP. Our findings highlight the importance of the allosteric role of ssRNA on the modulation of ATP hydrolysis and energy utilization.

Wolbachia bacteria, inherited through the female germ line, infect a large fraction of arthropod species. Many Wolbachia strains manipulate host reproduction, most commonly through cytoplasmic incompatibility (CI). CI, a conditional male sterility, results when Wolbachia-infected male insects mate with uninfected females; viability is restored if the female is similarly infected (called "rescue"). CI is used to help control mosquito-borne viruses such as dengue and Zika, but its mechanisms remain unknown. The coexpressed CI factors CifA and CifB form stable complexes in vitro, but the timing and function of this interaction in the insect are unresolved. CifA expression in the female germ line is sufficient for rescue. We report high-resolution structures of a CI-factor complex, CinA-CinB, which utilizes a unique binding mode between the CinA rescue factor and the CinB nuclease; the structures were validated by biochemical and yeast growth analyses. Importantly, transgenic expression in Drosophila of a nonbinding CinA mutant, designed based on the CinA-CinB structure, suggests CinA expressed in females must bind CinB imported by sperm in order to rescue embryonic viability. Binding between cognate factors is conserved in an enzymatically distinct CI system, CidA-CidB, suggesting universal features in Wolbachia CI induction and rescue.

氮化硼量子点是一类新型的零维纳米材料,它具备独特的荧光性能、高导热性、化学稳定性以及良好的生物相容性等出色的理化特性.氮化硼量子点在光电子学、电子元件、传感和催化、生物传感器和生物成像等领域已展现出极为广阔的应用前景,但其高效制备依然面临巨大挑战.因此,探索氮化硼量子点高效制备的新方法并进一步拓展其应用领域是当前纳米材料工程领域新的研究焦点.目前,氮化硼量子点的制备可以归纳为"自上而下"和"自下而上"两种方法.其中自上而下法是当前制备的主要方法,此方法类型多样,包括水热法、溶剂热法、超声剥离法以及碱金属插层法等.虽然自上而下法可实现大规模制备氮化硼量子点,但这类方法大都需要有机溶剂、强碱和高温等条件.自下而上的方法利用含有氮和硼元素的前体分子合成氮化硼量子点,可以实现量子点结构的精确控制,但这类方法不适合于大规模制备硼量子点.目前氮化硼量子点的大规模高效制备研究虽然还处在早期阶段,但已有的研究成果为其今后高效大规模的可控制备奠定了基础.同时,以量子点制备工作为基础,许多氮化硼量子点的应用工作也在如火如荼地开展之中,其中包括细胞成像、纤维染色、金属离子检测、化学发光传感器、指纹荧光成像和维生素的检测等,这些应用研究反过来也进一步促进了量子点制备工作的开展.本文对近年来氮化硼量子点的制备进行了系统的梳理,并对其在应用领域取得的研究成果进行了总结分析,最后提出了氮化硼量子点的制备及应用研究的思路,以期为后续的研究提供参考.

疏水蛋白(Hydrophobin)是具有表面活性的小分子量蛋白质,可以在界面自组装形成双亲性蛋白膜,从而改变界面亲疏水性.研究表明疏水蛋白无毒性且无免疫原性,基于这样的性质,疏水蛋白可用于材料表面修饰、食品塑形剂、纳米药物载体而进行靶向运输或生物传感器的信号精确识别等.近年来,在枯草芽孢杆菌生物被膜中发现了一种分泌型小分子量疏水蛋白BslA(原名YuaB).研究表明,枯草芽孢杆菌疏水蛋白BslA表达产量高,纯化过程简单、易于操作,可实现大规模生产,因而BslA具有更大的应用优势和开发价值.本文总结了BslA的性质、功能、结构等方面的信息,并与真菌疏水蛋白进行了比较分析,系统分析了其结构特点及应用价值.

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP51-486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP51-486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.

Enzymatic hydrolysis of polyethylene terephthalate (PET) is considered to be an environmentally friendly method for the recycling of plastic waste. Recently, a bacterial enzyme named IsPETase was found in Ideonella sakaiensis with the ability to degrade amorphous PET at ambient temperature suggesting its possible use in recycling of PET. However, applying the purified IsPETase in large-scale PET recycling has limitations, i.e., a complicated production process, high cost of single-use, and instability of the enzyme. Yeast cell surface display has proven to be an effectual alternative for improving enzyme degradation efficiency and realizing industrial applications. This chapter deals with the construction and application of a whole-cell biocatalyst by displaying IsPETase on the surface of yeast (Pichia pastoris) cells.