Analysis of the impact of temperature on the spectral shift in ultraviolet hyperspectral imaging spectrometers.

The imaging spectrometer's high performance in practical applications may be compromised by environmental factors, particularly temperature variations, posing a challenge to its stability. Temperature fluctuations can induce spectral shift, directly impacting the accuracy of spectral measurements, subsequently influencing the precision of radiometric measurements. To address this issue, this study investigates a dual-channel UV imaging spectrometer. This instrument boasts a wavelength calibration accuracy of 0.01 nm. This paper conducts an in-depth analysis of the various mechanisms through which temperature changes influence the spectral line offset in the imaging spectrometer, integrating actual orbital temperature data to discuss the instrument's temperature load settings. The impact of temperature on spectral shift is examined using finite element analysis and optical design software. Estimations of spectral shift were made based on temperature variations. Simulation results indicated that the maximum deviation of spectral shift is estimated at 0.018 nm under a temperature condition of 16 ± 1°C. Under a more controlled orbital temperature condition (16 ± 0.3°C), the maximum deviation of spectral shift decreased to 0.01 nm. Experimental data revealed that at 16 ± 1°C, the maximum deviation of spectral shift did not exceed 0.01 nm. This effectively corroborates our theoretical analysis. The relationship between temperature and spectral shift offers a crucial theoretical foundation for calibrating spectral measurements and managing the thermal conditions of the instrument.

Wzb of Vibrio vulnificus represents a new group of low-molecular-weight protein tyrosine phosphatases with a unique insertion in the W-loop.

Protein tyrosine phosphorylation regulates the production of capsular polysaccharide, an essential virulence factor of the deadly pathogen Vibrio vulnificus. The process requires the protein tyrosine kinase Wzc and its cognate phosphatase Wzb, both of which are largely uncharacterized. Herein, we report the structures of Wzb of V. vulnificus (VvWzb) in free and ligand-bound forms. VvWzb belongs to the low-molecular-weight protein tyrosine phosphatase (LMWPTP) family. Interestingly, it contains an extra four-residue insertion in the W-loop, distinct from all known LMWPTPs. The W-loop of VvWzb protrudes from the protein body in the free structure, but undergoes significant conformational changes to fold toward the active site upon ligand binding. Deleting the four-residue insertion from the W-loop severely impaired the enzymatic activity of VvWzb, indicating its importance for optimal catalysis. However, mutating individual residues or even substituting the whole insertion with four alanine residues only modestly decreased the enzymatic activity, suggesting that the contribution of the insertion to catalysis is not determined by the sequence specificity. Furthermore, inserting the four residues into Escherichia coli Wzb at the corresponding position enhanced its activity as well, indicating that the four-residue insertion in the W-loop can act as a general activity enhancing element for other LMWPTPs. The novel W-loop type and phylogenetic analysis suggested that VvWzb and its homologs should be classified into a new group of LMWPTPs. Our study sheds new insight into the catalytic mechanism and structural diversity of the LMWPTP family and promotes the understanding of the protein tyrosine phosphorylation system in prokaryotes.

Structural analysis of a Vibrio phospholipase reveals an unusual Ser-His-chloride catalytic triad.

Phospholipases can disrupt host membranes and are important virulence factors in many pathogens. VvPlpA is a phospholipase A2 secreted by Vibrio vulnificus and essential for virulence. Its homologs, termed thermolabile hemolysins (TLHs), are widely distributed in Vibrio bacteria, but no structural information for this virulence factor class is available. Herein, we report the crystal structure of VvPlpA to 1.4-Å resolution, revealing that VvPlpA contains an N-terminal domain of unknown function and a C-terminal phospholipase domain and that these two domains are packed closely together. The phospholipase domain adopts a typical SGNH hydrolase fold, containing the four conserved catalytic residues Ser, Gly, Asn, and His. Interestingly, the structure also disclosed that the phospholipase domain accommodates a chloride ion near the catalytic His residue. The chloride is five-coordinated in a distorted bipyramid geometry, accepting hydrogen bonds from a water molecule and the amino groups of surrounding residues. This chloride substitutes for the most common Asp/Glu residue and forms an unusual Ser-His-chloride catalytic triad in VvPlpA. The chloride may orient the catalytic His and stabilize the charge on its imidazole ring during catalysis. Indeed, VvPlpA activity depended on chloride concentration, confirming the important role of chloride in catalysis. The VvPlpA structure also revealed a large hydrophobic substrate-binding pocket that is capable of accommodating a long-chain acyl group. Our results provide the first structure of the TLH family and uncover an unusual Ser-His-chloride catalytic triad, expanding our knowledge on the biological role of chloride.

The dUTPase of white spot syndrome virus assembles its active sites in a noncanonical manner.

dUTPases are essential enzymes for maintaining genome integrity and have recently been shown to play moonlighting roles when containing extra sequences. Interestingly, the trimeric dUTPase of white spot syndrome virus (wDUT) harbors a sequence insert at the position preceding the C-terminal catalytic motif V (pre-V insert), rarely seen in other dUTPases. However, whether this extra sequence endows wDUT with additional properties is unknown. Herein, we present the crystal structures of wDUT in both ligand-free and ligand-bound forms. We observed that the pre-V insert in wDUT forms an unusual ß-hairpin structure in the domain-swapping region and thereby facilitates a unique orientation of the adjacent C-terminal segment, positioning the catalytic motif V onto the active site of its own subunit instead of a third subunit. Consequently, wDUT employs two-subunit active sites, unlike the widely accepted paradigm that the active site of trimeric dUTPase is contributed by all three subunits. According to results from local structural comparisons, the active-site configuration of wDUT is similar to that of known dUTPases. However, we also found that residues in the second-shell region of the active site are reconfigured in wDUT as an adaption to its unique C-terminal orientation. We also show that deletion of the pre-V insert significantly reduces wDUT's enzymatic activity and thermal stability. We hypothesize that this rare structural arrangement confers additional functionality to wDUT. In conclusion, our study expands the structural diversity in the conserved dUTPase family and illustrates how sequence insertion and amino acid substitution drive protein evolution cooperatively.

Structural basis for specific calcium binding by the polycystic-kidney-disease domain of Vibrio anguillarum protease Epp.

Extracellular proteases are often produced as pre-pro-enzyme and then undergo multiple processing steps to mature into the active form. The protease Epp, a virulent factor of Vibrio anguillarum, belongs to this family. Its maturation might be regulated by Ca2+ via its polycystic kidney disease (PKD) domain, but the molecular mechanism is unknown. Herein, we report the crystal structure of the first PKD domain from V. anguillarum Epp (Epp-PKD1) and its specific Ca2+-binding capacity. Epp-PKD1 exists as a monomer, consisting of seven ß-strands which form two ß-sheets stacking with each other. One Ca2+ is bound by the residues Asn3, Gln4, Asp27, Asp29, Asp68 and a water molecule with a pentagonal bipyramidal geometry. Incubating the apo Epp-PKD1 with Ca2+ but not Mg2+, Mn2+, or Zn2+, enhances the thermal and chemical stability of Epp-PKD1, indicating its specific binding to Ca2+. Epp-PKD1 shares high similarity in both sequence and overall structure with that of Vibrio cholerae PrtV, a homologous protease of Epp, however, they differ in the oligomeric state and local structure at the Ca2+-binding site, suggesting maturation of PrtV and Epp might be differently regulated by Ca2+. Likely, proteases may take advantage of the structural diversity in PKD domains to tune their Ca2+-regulated maturation process.

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination.

Severe acute respiratory syndrome (SARS) is a respiratory disease, caused by a coronavirus (SARS-CoV), that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I·C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by overexpression of the full-length N protein but not N amino acids 1 to 361, which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein.IMPORTANCE The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the Middle East respiratory syndrome CoV (MERS-CoV) N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response.

Global regulation of alternative RNA splicing by the SR-rich protein RBM39.

BACKGROUND: RBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. METHODS: In this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. RESULTS: RBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5' and 3' splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. CONCLUSIONS: RBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. GENERAL SIGNIFICANCE: The current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels.

Crystal structures of the isochorismatase domains from Vibrio anguillarum.

Antibiotic resistance is becoming a global threat and overuse of antibiotics in aquaculture disease control worsens the situation. To reduce the risk of drug resistance developed in aquaculture, safer biocontrol programs are needed. Antivirulence therapy, with less chance for developing drug resistance, is a promising approach. To facilitate antivirulence inhibitor design against Vibrio anguillarum, a serious aquaculture pathogen, we present crystal structures for isochorismatase domains of AngB and VabB, which are required to synthesize siderophore, a critical virulence factor. Both structures are highly similar to known isochorismatases in fold and active site, therefore we conclude inhibitors for isochorismatases can be developed in a common framework. The structural information will improve design of virulence inhibitors against Vibrio anguillarum. We also firstly report that isochorismatase family could bind endogenous metabolite during the hetero-expression process, which is likely nicotinic acid, nicotinamide or pyrazinic acid, based on structural analysis and affinity prediction. Taken together, our results provide precise structural information of isochorismatase domains for antivirulence inhibitor design against Vibrio anguillarum.

Malonyl-CoA pathway: a promising route for 3-hydroxypropionate biosynthesis.

3-Hydroxypropionate (3HP) is an attractive platform chemical, serving as a precursor to a variety of commodity chemicals like acrylate and acrylamide, as well as a monomer of a biodegradable plastic. To establish a sustainable way to produce these commercially important chemicals and materials, fermentative production of 3HP is widely investigated in recent years. It is reported that 3HP can be produced from several intermediates, such as glycerol, malonyl-CoA, and ß-alanine. Among all these biosynthetic routes, the malonyl-CoA pathway has some distinct advantages, including a broad feedstock spectrum, thermodynamic feasibility, and redox neutrality. To date, this pathway has been successfully constructed in various species including Escherichia coli, yeast and cyanobacteria, and optimized through carbon flux redirection, enzyme screening and engineering, and an increasing supply of energy and cofactors, resulting in significantly enhanced 3HP titer up to 40 g/L. These results show the feasibility of commercial manufacturing of 3HP and its derivatives in the future.

Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-ß. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRß in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei.

L-serine ammonia-lyase, as a member of the ß-family of pyridoxal-5'-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic ß-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/ß structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0-9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of ß-elimination enzymes and will provide a basis for further enzyme engineering.

miR-526a regulates apoptotic cell growth in human carcinoma cells.

MicroRNAs (miRNAs) play vital roles in the regulation of cell cycle, cell growth, apoptosis, and tumorigenesis. Our previous studies showed that miR-526a positively regulated innate immune response by suppressing CYLD expression, however, the functional relevance of miR-526a expression and cell growth remains to be evaluated. In this study, miR-526a overexpression was found to promote cancer cell proliferation, migration, and anchor-independent colony formation. The molecular mechanism(s) of miR-526a-mediated growth stimulation is associated with rapid cell cycle progression and inhibition of cell apoptosis by targeting CYLD. Taken together, these results provide evidence to show the stimulatory role of miR-526a in tumor migration and invasion through modulation of the canonical NF-κB signaling pathway.

Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity.

Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His(6)-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the α-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an α/ß domain and a long-α-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His(6) tag. Remarkably, the same protein harboring the sequence GPLG instead of the His(6) tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.

Molecular basis for SH3 domain regulation of F-BAR-mediated membrane deformation.

Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, alpha-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.

Catalytic site flexibility facilitates the substrate and catalytic promiscuity of Vibrio dual lipase/transferase.

Although enzyme catalysis is typified by high specificity, enzymes can catalyze various substrates (substrate promiscuity) and/or different reaction types (catalytic promiscuity) using a single active site. This interesting phenomenon is widely distributed in enzyme catalysis, with both fundamental and applied importance. To date, the mechanistic understanding of enzyme promiscuity is very limited. Herein, we report the structural mechanism underlying the substrate and catalytic promiscuity of Vibrio dual lipase/transferase (VDLT). Crystal structures of the VDLT from Vibrio alginolyticus (ValDLT) and its fatty acid complexes were solved, revealing prominent structural flexibility. In particular, the "Ser-His-Asp" catalytic triad machinery of ValDLT contains an intrinsically flexible oxyanion hole. Analysis of ligand-bound structures and mutagenesis showed that the flexible oxyanion hole and other binding residues can undergo distinct conformational changes to facilitate substrate and catalytic promiscuity. Our study reveals a previously unknown flexible form of the famous catalytic triad machinery and proposes a "catalytic site tuning" mechanism to expand the mechanistic paradigm of enzyme promiscuity.

Lysine acetylation of Escherichia coli lactate dehydrogenase regulates enzyme activity and lactate synthesis.

As an evolutionarily conserved posttranslational modification, protein lysine acetylation plays important roles in many physiological and metabolic processes. However, there are few reports about the applications of lysine acetylation in metabolic regulations. Lactate is a main byproduct in microbial fermentation, and itself also an important bulk chemical with considerable commercial values in many fields. Lactate dehydrogenase (LdhA) is the key enzyme catalyzing lactate synthesis from pyruvate. Here, we reported that Escherichia coli LdhA can be acetylated and the acetylated lysine sites were identified by mass spectrometry. The effects and regulatory mechanisms of acetylated sites on LdhA activity were characterized. Finally, lysine acetylation was successfully used to regulate the lactate synthesis. LdhA (K9R) mutant overexpressed strain improved the lactate titer and glucose conversion efficiency by 1.74 folds than that of wild-type LdhA overexpressed strain. LdhA (K154Q-K248Q) mutant can inhibit lactate accumulation and improve 3HP production. Our study established a paradigm for lysine acetylation in lactate synthesis regulation and suggested that lysine acetylation may be a promising strategy to improve the target production and conversion efficiency in microbial synthesis. The application of lysine acetylation in regulating lactate synthesis also provides a reference for the treatment of lactate-related diseases.

Highly pathogenic coronavirus N protein aggravates inflammation by MASP-2-mediated lectin complement pathway overactivation.

Excessive inflammatory responses contribute to the pathogenesis and lethality of highly pathogenic human coronaviruses, but the underlying mechanism remains unclear. In this study, the N proteins of highly pathogenic human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were found to bind MASP-2, a key serine protease in the lectin pathway of complement activation, resulting in excessive complement activation by potentiating MBL-dependent MASP-2 activation, and the deposition of MASP-2, C4b, activated C3 and C5b-9. Aggravated inflammatory lung injury was observed in mice infected with adenovirus expressing the N protein. Complement hyperactivation was also observed in SARS-CoV-2-infected patients. Either blocking the N protein:MASP-2 interaction, MASP-2 depletion or suppressing complement activation can significantly alleviate N protein-induced complement hyperactivation and lung injury in vitro and in vivo. Altogether, these data suggested that complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.

Association of elevated HIF-2α levels with low Beclin 1 expression and poor prognosis in patients with chondrosarcoma.

BACKGROUND: Hypoxia inducible factor (HIF)-2α is an important transcription factor that contributes to tumor proliferation and progression. Beclin 1 is a key mediator of autophagy, and dysfunction of Beclin 1 is implicated in tumorigenicity. This study was designed to investigate the expression patterns of HIF-2α and Beclin 1 and to clarify their clinical significance in chondrosarcoma. METHODS: The mRNA and protein levels of HIF-2α and Beclin 1 in chondrosarcoma and the corresponding nontumor tissues were analyzed by real-time polymerase chain reaction and Western blot, respectively. The protein expression of HIF-2α and Beclin 1 was investigated by immunohistochemistry, and their associations with clinicopathological factors and overall survival were evaluated. RESULTS: HIF-2α was remarkably elevated, whereas Beclin 1 was significantly reduced in chondrosarcoma compared with the corresponding nontumor tissues. High HIF-2α level and negative Beclin 1 expression were 52.9% and 58.8% in chondrosarcoma specimens, respectively. HIF-2α and Beclin 1 were associated with histological grade and Musculoskeletal Tumor Society stage. There was a significant inverse relationship between HIF-2α and Beclin 1. HIF-2α and Beclin 1 had significant impacts on the prognosis of chondrosarcoma patients. Multivariate analysis revealed that Beclin 1 was an independent prognostic factor for overall survival of patients with chondrosarcoma. CONCLUSIONS: Elevated HIF-2α levels associated with low Beclin 1 expression play a role in the development of chondrosarcoma. Beclin 1 can serve as a novel biomarker to predict survival of chondrosarcoma patients, and may represent a potential therapeutic target.

Structural basis of staphylococcal Stl inhibition on a eukaryotic dUTPase.

dUTPases are key enzymes in all life kingdoms. A staphylococcal repressor protein (Stl) inhibited dUTPases from multiple species to various extents. Understanding the molecular basis underlying the inhibition differences is crucial to develop effective proteinaceous inhibitors of dUTPases. Herein, we report the complex structure of Stl N-terminal domain (StlN-ter) and Litopenaeus vannamei dUTPase domain (lvDUT65-210). Stl inhibited lvDUT65-210 through its N-terminal domain. The lvDUT65-210-StlN-ter complex structure revealed a heterohexamer encompassing three StlN-ter monomers bound to one lvDUT65-210 trimer, generating two types of Stl-dUTPase interfaces. Interface I is formed by Stl interaction with the lvDUT65-210 active-site region that is contributed by motifs I-IV from its two subunits; interface II results from Stl binding to the C-terminal motif V of the third lvDUT65-210 subunit. Structural comparison revealed both conserved features and obvious differences in Stl-dUTPase interaction patterns, giving clues about the inhibition differences of Stl on dUTPases. Noticeably, interface II is only observed in lvDUT65-210-StlN-ter. The Stl-interacting residues of lvDUT65-210 are conserved in other eukaryotic dUTPases, particularly human dUTPase. Altogether, our study presents the first structural model of Stl interaction with eukaryotic dUTPase, contributing to a more complete view of Stl inhibition and facilitating the development of proteinaceous inhibitor for eukaryotic dUTPases.