Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Targeting proto-oncogene B-MYB G-quadruplex with a nucleic acid-based fluorescent probe.

The B-MYB gene encodes a transcription factor (B-MYB) that regulates cell growth and survival. Abnormal expression of B-MYB is frequently observed in lung cancer and poses challenges for targeted drug therapy. Oncogenes often contain DNA structures called G-quadruplexes (G4s) in their promoter regions, and B-MYB is no exception. These G4s play roles in genetic regulation and are potential cancer treatment targets. In this study, a probe was designed to specifically identify a G4 within the promoter region of the B-MYB gene. This probe combines an acridine derivative ligand with a DNA segment complementary to the target sequence, enabling it to hybridize with the adjacent sequence of the G4 being investigated. Biophysical studies demonstrated that the acridine derivative ligands C5NH2 and C8NH2 not only effectively stabilized the G4 structure but also exhibited moderate affinity. They were capable of altering the G4 topology and exhibited enhanced fluorescence emission in the presence of this quadruplex. Additionally, these ligands increased the number of G4s observed in cellular studies. Through various biophysical studies, the target sequence was shown to form a G4 structure, even with an extra nucleotide tail added to its flanking region. Cellular studies confirmed the co-localization between the target sequence and the developed probe.

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

Electron microscopy mapping of the DNA-binding sites of monomeric, dimeric, and multimeric KSHV RTA protein.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus associated with several human cancers, typically in patients with compromised immune systems. Herpesviruses establish lifelong infections in hosts in part due to the two phases of infection: the dormant and active phases. Effective antiviral treatments to prevent the production of new viruses are needed to treat KSHV. A detailed microscopy-based investigation of the molecular interactions between viral protein and viral DNA revealed how protein-protein interactions play a role in DNA-binding specificity. This analysis will lead to a more in-depth understanding of KSHV DNA replication and serve as the basis for anti-viral therapies that disrupt and prevent the protein-DNA interactions, thereby decreasing spread to new hosts.

Synergistic Binding of ATP and Nucleic Acids Necessitates UPF1's ATPase Functional Cycle.

UPF1 is a core protein in the nonsense mRNA degradation (NMD) surveillance pathway that degrades aberrant mRNA. UPF1 has both ATPase and RNA helicase activities, but it exhibits mutually exclusive binding of ATP and RNA. This suggests intricate allosteric coupling between ATP and RNA binding that remains unresolved. In this study, we used molecular dynamics simulations and dynamic network analyses to probe the dynamics and free energy landscapes covering UPF1 crystal structures resolved in the Apo state, the ATP bound state, and the ATP-RNA bound (catalytic transition) state. Free energy calculations show that in the presence of ATP and RNA, the transition from the Apo state to the ATP bound state is an uphill process but becomes a downhill process when transitioning to the catalytic transition state. Allostery potential analyses reveal that the Apo and catalytic transition states are mutually allosterically activated toward each other, reflecting the intrinsic ATPase function of UPF1. The Apo state is also allosterically activated toward the ATP bound state. However, binding ATP alone leads to an allosterically trapped state that is difficult to revert to either the Apo or the catalytic transition state. The high allostery potential of Apo UPF1 toward different states results in a "first come, first served" mechanism that requires the synergistic binding of ATP and RNA to drive the ATPase cycle. Our results reconcile UPF1's ATPase and RNA helicase activities within an allostery framework and may apply to other SF1 helicases, as we demonstrate that UPF1's allostery signaling pathways prefer the RecA1 domain over the equally fold-conserved RecA2 domain, and this preference coincides with higher sequence conservation in the RecA1 domain across typical human SF1 helicases.

In silico study reveals unconventional interactions between MDC1 of DDR and Beclin-1 of autophagy.

DNA damage response (DDR) and autophagy are concerned with maintaining cellular homeostasis and dysregulation of these two pathways lead to pathologic conditions including tumorigenesis. Autophagy is activated as a protective mechanism during DDR which is indicative of their functional cooperativity but the molecular mechanism leading to the convergence of these two pathways during genotoxic stress remains elusive. In this study, through in silico analysis, we have shown an interaction between the Mediator of DNA damage checkpoint 1 (MDC1), an important DDR-associated protein, and Beclin-1, an autophagy inducer. MDC1 is an adaptor or scaffold protein known to regulate DDR, apoptosis, and cell cycle progression. While, Beclin-1 is involved in autophagosome nucleation and exhibits affinity for binding to Fork-head-associated domain (FHA) containing proteins. The FHA domain is commonly conserved in DDR-related proteins including MDC1. Through molecular docking, we have predicted the modeled complex between the MDC1 FHA domain and the Beclin-1 Coiled coil domain (CCD). The docking complex was modeled using ClusPro2.0, based on the crystal structure for the dimerized MDC1 FHA domain and Beclin-1 CCD. The complex stability and binding affinities were assessed using a Ramachandran plot, MD simulation, MM/GBSA, and PRODIGY webserver. Finally, the hot-spot residues at the interface were determined using computational alanine scanning by the DrugScorePPI webserver. Our analysis unveils significant interaction between MDC1 and Beclin-1, involving hydrogen bonds, non-bonded contacts, and salt bridges and indicates MDC1 possibly recruits Beclin-1 to the DSBs, as a consequence of which Beclin-1 is able to modulate DDR.

Computational investigations on the potential role of hygrophorones as quorum sensing inhibitors against LasR protein of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram negative, rod shape bacterium that infects people with compromised immune systems, such as those suffering from AIDS, organ transplantation and cancer. This bacterium is responsible for diseases like cystic fibrosis, chronic lung infection, and ulcerative keratitis. It is diagnosed in most of the patients who were on prolonged ventilation with long term critical care stay. P. aeruginosa develops rapid antimicrobial resistance that is challenging for the treatment and eventually it causes high mortality rate. Thus, the search for potential novel inhibitors that can inhibit the pathogenic activity of P. aeruginosa is of utmost importance. In P. aeruginosa, an important protein, LasR that participates in the gene regulations and expressions has been proposed to be a suitable drug target. Here, we identify a set of hygrophorone molecules as effective inhibitors for this LasR protein based on molecular docking and simulations studies. At first, large number of hygrophorone series of small molecules were screened against the LasR protein and their binding affinities were assessed based on the docking scores. Top scored molecules were selected for calculating various pharmacophore properties, and finally, their potential in inhibiting the LasR protein was delineated by atomistic molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area-based calculations. Both docking and simulations studies reveal that a subset of hygrophorone molecules have a good binding affinity for LasR protein and form stable LasR-inhibitor complexes. The present study illustrates that the hygrophorones can be effective inhibitors for the LasR protein and will spur further in vitro studies that would aid to the ongoing search for new antibiotics.Communicated by Ramaswamy H. Sarma.

Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery.

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric linker." While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

Cyclin-dependent kinase-mediated phosphorylation and the negative regulatory domain of transcription factor B-Myb modulate its DNA binding.

B-Myb is a highly conserved member of the vertebrate Myb family of transcription factors that plays a critical role in cell-cycle progression and proliferation. Myb proteins activate Myb-dependent promoters by interacting specifically with Myb-binding site (MBS) sequences using their DNA-binding domain (DBD). Transactivation of MBS promoters by B-Myb is repressed by its negative regulatory domain (NRD), and phosphorylation of the NRD by Cdk2-CyclinA relieves the repression to activate B-Myb-dependent promoters. However, the structural mechanisms underlying autoinhibition and activation of B-Myb-mediated transcription have been poorly characterized. Here, we determined that a region in the B-Myb NRD (residues 510-600) directly associates with the DBD and inhibits binding of the DBD to the MBS DNA sequence. We demonstrate using biophysical assays that phosphorylation of the NRD at T515, T518, and T520 is sufficient to disrupt the interaction between the NRD and the DBD, which results in increased affinity for MBS DNA and increased B-Myb-dependent promoter activation in cell assays. Our biochemical characterization of B-Myb autoregulation and the activating effects of phosphorylation provide insight into how B-Myb functions as a site-specific transcription factor.

OFF-switching property of quorum sensor LuxR via As(III)-induced insoluble form.

Whole-cell sensors for arsenite detection have been developed exclusively based on the natural arsenite (As(III)) sensory protein ArsR for arsenic metabolism. This study reports that the quorum-sensing LuxR/Plux system from Vibrio fischeri, which is completely unrelated to arsenic metabolism, responds to As(III) in a dose-dependent manner. Due to as many as 9 cysteine residues, which has a high binding affinity with As(III), LuxR underwent As(III)-induced insoluble form, thereby reducing its effective cellular concentration. Accordingly, the expression level of green fluorescent protein under the control of Plux gradually decreased with increasing As(III) concentration in the medium. This is a novel As(III)-detection system that has never been proposed before, with a unique ON-to-OFF transfer function.

The Role of Charge Density Coupled DNA Bending in Transcription Factor Sequence Binding Specificity: A Generic Mechanism for Indirect Readout.

The accurate reading of genetic information during transcription is essential for the expression of genes. Sequence binding specificity very often is attributed to short-range, usually specific interactions between amino acid residues and individual nucleotide bases through hydrogen bonding or hydrophobic contacts: "base readout" (direct readout). In contrast, many proteins recognize DNA sequences in an alternative fashion via "shape readout" (indirect readout), where many elements of the DNA sequence cooperate to localize the transcription factor. In this study, we use a coarse-grained protein-DNA model to investigate the origin of the sequence specificity of the protein PU.1 binding to its binding sites for a series of DNA sequences. We find that the binding specificity of PU.1 is achieved primarily via a nonspecific electrostatically driven DNA mechanism involving the change in the elastic properties of the DNA. To understand the underlying mechanism, we analyze how the mechanical properties of DNA change in relation to the location of the PU.1 bound along DNA. The simulations first show that electrostatic interactions between PU.1 and DNA can cause complex DNA conformational/dynamics changes. Using a semiflexible polymer theory, we find that PU.1 influences the DNA dynamics through a second-order mechanical effect. When PU.1 binds nonspecifically to the DNA via electrostatics, the DNA becomes stiffer and the protein slides along DNA in a search mode. In contrast, once the protein finds its specific binding site, the DNA becomes softer there. PU.1 thus locks into place through configurational entropy effects, which we suggest is a generic mechanism for indirect readout.

Multivalency enables unidirectional switch-like competition between intrinsically disordered proteins.

Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ϕC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell.

Structural basis of branch site recognition by the human spliceosome.

Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17S U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)­dependent remodeling and binding to the pre­messenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.

O-GlcNAc modified-TIP60/KAT5 is required for PCK1 deficiency-induced HCC metastasis.

Aberrant glucose metabolism and elevated O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) are hallmarks of hepatocellular carcinoma (HCC). Loss of phosphoenolpyruvate carboxykinase 1 (PCK1), the major rate-limiting enzyme of hepatic gluconeogenesis, increases hexosamine biosynthetic pathway (HBP)-mediated protein O-GlcNAcylation in hepatoma cell and promotes cell growth and proliferation. However, whether PCK1 deficiency and hyper O-GlcNAcylation can induce HCC metastasis is largely unknown. Here, gain- and loss-of-function studies demonstrate that PCK1 suppresses HCC metastasis in vitro and in vivo. Specifically, lysine acetyltransferase 5 (KAT5), belonging to the MYST family of histone acetyltransferases (HAT), is highly modified by O-GlcNAcylation in PCK1 knockout hepatoma cells. Mechanistically, PCK1 depletion suppressed KAT5 ubiquitination by increasing its O-GlcNAcylation, thereby stabilizing KAT5. KAT5 O-GlcNAcylation epigenetically activates TWIST1 expression via histone H4 acetylation, and enhances MMP9 and MMP14 expression via c-Myc acetylation, thus promoting epithelial-mesenchymal transition (EMT) in HCC. In addition, targeting HBP-mediated O-GlcNAcylation of KAT5 inhibits lung metastasis of HCC in hepatospecific Pck1-deletion mice. Collectively, our findings demonstrate that PCK1 depletion increases O-GlcNAcylation of KAT5, epigenetically induces TWIST1 expression and promotes HCC metastasis, and link metabolic enzyme, post-translational modification (PTM) with epigenetic regulation.

Molecular Mechanisms and Animal Models of HBV-Related Hepatocellular Carcinoma: With Emphasis on Metastatic Tumor Antigen 1.

Hepatocellular carcinoma (HCC) is an important cause of cancer death worldwide, and hepatitis B virus (HBV) infection is a major etiology, particularly in the Asia-Pacific region. Lack of sensitive biomarkers for early diagnosis of HCC and lack of effective therapeutics for patients with advanced HCC are the main reasons for high HCC mortality; these clinical needs are linked to the molecular heterogeneity of hepatocarcinogenesis. Animal models are the basis of preclinical and translational research in HBV-related HCC (HBV-HCC). Recent advances in methodology have allowed the development of several animal models to address various aspects of chronic liver disease, including HCC, which HBV causes in humans. Currently, multiple HBV-HCC animal models, including conventional, hydrodynamics-transfection-based, viral vector-mediated transgenic, and xenograft mice models, as well as the hepadnavirus-infected tree shrew and woodchuck models, are available. This review provides an overview of molecular mechanisms and animal models of HBV-HCC. Additionally, the metastatic tumor antigen 1 (MTA1), a cancer-promoting molecule, was introduced as an example to address the importance of a suitable animal model for studying HBV-related hepatocarcinogenesis.

Proteasomal activator 28 gamma stabilizes hepatitis B virus X protein by competitively inhibiting the Siah-1-mediated proteasomal degradation.

Proteasomal activator 28 gamma (PA28γ) upregulates the levels of HBx, a regulatory protein of hepatitis B virus (HBV) to stimulate HBV replication; however, the detailed mechanism remains unknown. Here, we found that PA28γ impaired the ability of seven in absentia homolog 1 (Siah-1) as an E3 ubiquitin ligase of HBx. PA28γ competitively inhibited the binding of Siah-1 to HBx in human hepatoma cells. Accordingly, PA28γ increased the stability of HBx and decreased HBx ubiquitination, abolishing the potential of Siah-1 to downregulate HBx levels. PA28γ also executed its role as an antagonist of Siah-1 during HBV replication, as demonstrated by an in vitro HBV replication system. The present study may provide insights into the mechanisms underlying the regulation of HBV replication.

Screening of the HBx transactivation domain interacting proteins and the function of interactor Pin1 in HBV replication.

Hepatitis B virus (HBV) X protein (HBx) has been determined to play a crucial role in the replication and transcription of HBV, and its biological functions mainly depend on the interaction with other host proteins. This study aims at screening the proteins that bind to the key functional domain of HBx by integrated proteomics. Proteins that specifically bind to the transactivation domain of HBx were selected by comparing interactors of full-length HBx and HBx-D5 truncation determined by glutathione-S-transferase (GST) pull-down assay combined with mass spectrometry (MS). The function of HBx interactor Pin1 in HBV replication was further investigated by in vitro experiments. In this study, a total of 189 proteins were identified from HepG2 cells that specifically bind to the transactivation domain of HBx by GST pull-down and subsequent MS. After gene ontology (GO) analysis, Pin1 was selected as the protein with the highest score in the largest cluster functioning in protein binding, and also classified into the cluster of proteins with the function of structural molecule activity, which is of great potential to be involved in HBV life cycle. The interaction between Pin1 and HBx has been further confirmed by Ni2+-NTA pulldown assay, co-immunoprecipitation, and immunofluorescence microscopy. HBsAg and HBeAg levels significantly decreased in Pin1 expression inhibited HepG2.2.15 cells. Besides, the inhibition of Pin1 expression in HepG2 cells impeded the restored replication of HBx-deficient HBV repaired by ectopic HBx expression. In conclusion, our study identified Pin1 as an interactor binds to the transactivation domain of HBx, and suggested the potential association between Pin1 and the function of HBx in HBV replication.

Long noncoding RNA IL6-AS1 is highly expressed in chronic obstructive pulmonary disease and is associated with interleukin 6 by targeting miR-149-5p and early B-cell factor 1.

Chronic obstructive pulmonary disease is a complex condition with multiple etiologies, including inflammation. We identified a novel long noncoding RNA (lncRNA), interleukin 6 antisense RNA 1 (IL6-AS1), which is upregulated in this disease and is associated with airway inflammation. We found that IL6-AS1 promotes the expression of inflammatory factors, especially interleukin (IL) 6. Mechanistically, cytoplasmic IL6-AS1 acts as an endogenous sponge by competitively binding to the microRNA miR-149-5p to stabilize IL-6 mRNA. Nuclear IL6-AS1 promotes IL-6 transcription by recruiting early B-cell factor 1 to the IL-6 promoter, which increases the methylation of the H3K4 histone and acetylation of the H3K27 histone. We propose a model of lncRNA expression in both the nucleus and cytoplasm that exerts similar effects through differing mechanisms, and IL6-AS1 probably increases inflammation via multiple pathways.

S-Adenosyl-l-ethionine is a Catalytically Competent Analog of S-Adenosyl-l-methione (SAM) in the Radical SAM Enzyme HydG.

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5'-deoxyadenosyl radical (5'-dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5'-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5'-dAdo•. Here we present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically-equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE bound to HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster, generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.

The structural mechanism for the nucleoside tri- and diphosphate hydrolysis activity of Ntdp from Staphylococcus aureus.

Staphylococcus aureus is a well-known clinical pathogenic bacterium. In recent years, due to the emergence of multiple drug-resistant strains of S. aureus in clinical practice, S. aureus infections have become an increasingly severe clinical problem. Ntdp (nucleoside tri- and diphosphatase, also known as Sa1684) is a nucleotide phosphatase that has a significant effect on the proliferation of S. aureus colonies and the killing ability of the host. Here, we identified the nucleoside tri- and diphosphate hydrolysis activity of Ntdp and obtained the three-dimensional structures of apo-Ntdp and three substrate analog (ATPγ S, GDPß S, and GTPγ S) complexes of Ntdp. Through structural analysis and biochemical verification, we illustrated the structural basis for the divalent cation selectivity, substrate recognition model, and catalytic mechanism of Ntdp. We also revealed a possible basal functional pattern of the DUF402 domain and hypothesized the potential pathways by which the protein regulates the expression of the two-component regulatory factor agr and the downstream virulence factors. Overall, the above findings provide crucial insights into our understanding of the Ntdp functional mechanism in the infection process.