Specific Protein-protein Interactions Research Articles

Unraveling the role of physicochemical differences in predicting protein-protein interactions.

The ability to accurately predict protein-protein interactions is critically important for understanding major cellular processes. However, current experimental and computational approaches for identifying them are technically very challenging and still have limited success. We propose a new computational method for predicting protein-protein interactions using only primary sequence information. It utilizes the concept of physicochemical similarity to determine which interactions will most likely occur. In our approach, the physicochemical features of proteins are extracted using bioinformatics tools for different organisms. Then they are utilized in a machine-learning method to identify successful protein-protein interactions via correlation analysis. It was found that the most important property that correlates most with the protein-protein interactions for all studied organisms is dipeptide amino acid composition (the frequency of specific amino acid pairs in a protein sequence). While current approaches often overlook the specificity of protein-protein interactions with different organisms, our method yields context-specific features that determine protein-protein interactions. The analysis is specifically applied to the bacterial two-component system that includes histidine kinase and transcriptional response regulators, as well as to the barnase-barstar complex, demonstrating the method's versatility across different biological systems. Our approach can be applied to predict protein-protein interactions in any biological system, providing an important tool for investigating complex biological processes' mechanisms.

The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8 to 10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine-binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.

Allosteric regulation of the tyrosine phosphatase PTP1B by a protein-protein interaction.

The rapid identification of protein-protein interactions has been significantly enabled by mass spectrometry (MS) proteomics-based methods, including affinity purification-MS, crosslinking-MS, and proximity-labeling proteomics. While these methods can reveal networks of interacting proteins, they cannot reveal how specific protein-protein interactions alter cell signaling or protein function. For instance, when two proteins interact, there can be emergent signaling processes driven purely by the individual activities of those proteins being co-localized. Alternatively, protein-protein interactions can allosterically regulate function, enhancing or suppressing activity in response to binding. In this work, we investigate the interaction between the tyrosine phosphatase PTP1B and the adaptor protein Grb2, which have been annotated as binding partners in a number of proteomics studies. This interaction has been postulated to co-localize PTP1B with its substrate IRS-1 by forming a ternary complex, thereby enhancing the dephosphorylation of IRS-1 to suppress insulin signaling. Here, we report that Grb2 binding to PTP1B also allosterically enhances PTP1B catalytic activity. We show that this interaction is dependent on the proline-rich region of PTP1B, which interacts with the C-terminal SH3 domain of Grb2. Using NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) we show that Grb2 binding alters PTP1B structure and/or dynamics. Finally, we use MS proteomics to identify other interactors of the PTP1B proline-rich region that may also regulate PTP1B function similarly to Grb2. This work presents one of the first examples of a protein allosterically regulating the enzymatic activity of PTP1B and lays the foundation for discovering new mechanisms of PTP1B regulation in cell signaling.

A Hitchhiker's Guide Through the Cell: The World According to the Capsids of Alphaherpesviruses.

The nucleoplasm, the cytosol, the inside of virions, and again the cytosol comprise the world in which the capsids of alphaherpesviruses encounter viral and host proteins that support or limit them in performing their tasks. Here, we review the fascinating conundrum of how specific protein-protein interactions late in alphaherpesvirus infection orchestrate capsid nuclear assembly, nuclear egress, and cytoplasmic envelopment, but target incoming capsids to the nuclear pores in naive cells to inject the viral genomes into the nucleoplasm for viral transcription and replication. Multiple capsid interactions with viral and host proteins have been characterized using viral mutants and assays that reconstitute key stages of the infection cycle. Keratinocytes, fibroblasts, mucosal epithelial cells, neurons, and immune cells employ cell type-specific intrinsic and cytokine-induced resistance mechanisms to restrict several stages of the viral infection cycle. However, concomitantly, alphaherpesviruses have evolved countermeasures to ensure efficient capsid function during infection.

Silver nanoparticles induce formation of multi-protein aggregates that contain cadherin but do not colocalize with nanoparticles

Silver nanoparticles (AgNPs) are increasingly incorporated in diverse products to confer antimicrobial properties. They are released into the environment during manufacture, after disposal, and from the products during use. Because AgNPs bioaccumulate in brain, it is important to understand how they interact with neural cell physiology. We found that the focal adhesion (FA)-associated protein cadherin aggregated in a dose-dependent response to AgNP exposure in differentiating cultured B35 neuroblastoma cells. These aggregates tended to colocalize with F-actin inclusions that form in response to AgNP and also contain β-catenin. However, using hyperspectral microscopy, we demonstrate that these multi-protein aggregates did not colocalize with the AgNPs themselves. Furthermore, expression and organization of the FA protein vinculin did not change in cells exposed to AgNP. Our findings suggest that AgNPs activate an intermediate mechanism which leads to formation of aggregates via specific protein-protein interactions. Finally, we detail the changes in hyperspectral profiles of AgNPs during different stages of cell culture and immunocytochemistry processing. AgNPs in citrate-stabilized solution present mostly blue with some rainbow spectra and these are maintained upon mounting in Prolong Gold. Exposure to tissue culture medium results in a uniform green spectral shift that is not further altered by fixation and protein block steps of immunocytochemistry.

Role of Mod(mdg4)-67.2 Protein in Interactions between Su(Hw)-Dependent Complexes and Their Recruitment to Chromatin.

Su(Hw) belongs to the class of proteins that organize chromosome architecture, determine promoter activity, and participate in formation of the boundaries/insulators between the regulatory domains. This protein contains a cluster of 12 zinc fingers of the C2H2 type, some of which are responsible for binding to the consensus site. The Su(Hw) protein forms complex with the Mod(mdg4)-67.2 and the CP190 proteins, where the last one binds to all known Drosophila insulators. To further study functioning of the Su(Hw)-dependent complexes, we used the previously described su(Hw)E8 mutation with inactive seventh zinc finger, which produces mutant protein that cannot bind to the consensus site. The present work shows that the Su(Hw)E8 protein continues to directly interact with the CP190 and Mod(mdg4)-67.2 proteins. Through interaction with Mod(mdg4)-67.2, the Su(Hw)E8 protein can be recruited into the Su(Hw)-dependent complexes formed on chromatin and enhance their insulator activity. Our results demonstrate that the Su(Hw) dependent complexes without bound DNA can be recruited to the Su(Hw) binding sites through the specific protein-protein interactions that are stabilized by Mod(mdg4)-67.2.

PPII-AEAT: Prediction of protein-protein interaction inhibitors based on autoencoders with adversarial training

Protein-protein interactions (PPIs) have shown increasing potential as novel drug targets. The design and development of small molecule inhibitors targeting specific PPIs are crucial for the prevention and treatment of related diseases. Accordingly, effective computational methods are highly desired to meet the emerging need for the large-scale accurate prediction of PPI inhibitors. However, existing machine learning models rely heavily on the manual screening of features and lack generalizability. Here, we propose a new PPI inhibitor prediction method based on autoencoders with adversarial training (named PPII-AEAT) that can adaptively learn molecule representation to cope with different PPI targets. First, Extended-connectivity fingerprints and Mordred descriptors are employed to extract the primary features of small molecular compounds. Then, an autoencoder architecture is trained in three phases to learn high-level representations and predict inhibitory scores. We evaluate PPII-AEAT on nine PPI targets and two different tasks, including the PPI inhibitor identification task and inhibitory potency prediction task. The experimental results show that our proposed PPII-AEAT outperforms state-of-the-art methods.

Role of tungsten disulfide quantum dots in specific protein-protein interactions at air-water interface.

The intriguing network of antibody-antigen (Ab-Ag) interactions is highly governed by environmental perturbations and the nature of biomolecular interaction. Protein-protein interactions (PPIs) have potential applications in developing protein-adsorption-based sensors and nano-scale materials. Therefore, characterizing PPIs in the presence of a nanomaterial at the molecular level becomes imperative. The present work involves the investigation of antiferritin-ferritin (Ab-Ag) protein interactions under the influence of tungsten disulfide quantum dots (WS2 QDs). Isothermal calorimetry and contact angle measurements validated the strong influence of WS2 QDs on Ab-Ag interactions. The interfacial signatures of nano-bio-interactions were evaluated using sum frequency generation vibration spectroscopy (SFG-VS) at the air-water interface. Our SFG results reveal a variation in the tilt angle of methyl groups by ∼12° ± 2° for the Ab-Ag system in the presence of WS2 QDs. The results illustrated an enhanced ordering of water molecules in the presence of QDs, which underpins the active role of interfacial water molecules during nano-bio-interactions. We have also witnessed a differential impact of QDs on Ab-Ag by raising the concentration of the Ab-Ag combination, which showcased an increased inter-molecular interaction among the Ab and Ag molecules and a minimal influence on the methyl tilt angle. These findings suggest the formation of stronger and ordered Ab-Ag complexes upon introducing WS2 QDs in the aqueous medium and signify the potentiality of WS2 QDs relevant to protein-based sensing assays.

Specific protein interactions between rice members of the GT43 and GT47 families form various central cores of putative xylan synthase complexes.

Members of the glycosyltransferase (GT)43 and GT47 families have been associated with heteroxylan synthesis in both dicots and monocots and are thought to assemble into central cores of putative xylan synthase complexes (XSCs). Currently, it is unknown whether protein-protein interactions within these central cores are specific, how many such complexes exist, and whether these complexes are functionally redundant. Here, we used gene association network and co-expression approaches in rice to identify four OsGT43s and four OsGT47s that assemble into different GT43/GT47 complexes. Using two independent methods, we showed that (i) these GTs assemble into at least six unique complexes through specific protein-protein interactions and (ii) the proteins interact directly in vitro. Confocal microscopy showed that, when alone, all OsGT43s were retained in the endoplasmic reticulum (ER), while all OsGT47s were localized in the Golgi. co-expression of OsGT43s and OsGT47s displayed complexes that form in the ER but accumulate in Golgi. ER-to-Golgi trafficking appears to require interactions between OsGT43s and OsGT47s. Comparison of the central cores of the three putative rice OsXSCs to wheat, asparagus, and Arabidopsis XSCs, showed great variation in GT43/GT47 combinations, which makes the identification of orthologous central cores between grasses and dicots challenging. However, the emerging picture is that all central cores from these species seem to have at least one member of the IRX10/IRX10-L clade in the GT47 family in common, suggesting greater functional importance for this family in xylan synthesis. Our findings provide a new framework for future investigation of heteroxylan biosynthesis and function in monocots.

Deciphering the molecular functionality of Cdc45 in replisomal complex

The members of DHH superfamily have been reported with diverse substrate spectrum and play pivotal roles in replication, repair, and RNA metabolism. This family comprises phosphatases, phosphoesterase and bifunctional enzymes having nanoRNase and phosphatase activities. Cell cycle factor Cdc45, a member of this superfamily, is crucial for movement of the replication fork during DNA replication and an important component of the replisome. The specific protein-protein interactions of Cdc45 with other factors along with helicase moderate the faithful DNA replication process. However, the exact biochemical functions of this factor are still unknown and need further investigation. Here, we studied the biochemical roles of Cdc45 and its molecular interactions within the replisomal complex. The alteration in the level of protein, observed when DNA damage is induced in-vivo, suggests its association with DNA replication stress. We analyzed protein Cdc45, providing new insights about the molecular and biochemical functionality of this replisomal factor.

Isolating Interaction-Null/Impaired Mutants using the Yeast Two-Hybrid Assay.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with thepotential to accelerate immunotherapy design.

Integrated epigenomic and network analysis identifies novel DNA methylation signature in atrial fibrillation

Abstract Background CD4+ T cells are associated with atrial fibrillation (AF) pathogenesis but mechanistic insights as well as epigenetic phenomena underlying the onset and progression of AF are not fully understood. Purpose To identify differential methylation signature in circulating CD4+ T lymphocytes isolated from AF patients and HS enrolled in the DIANA clinical trial through epigenome and network medicine approaches. Methods DNA was extracted from CD4+ T lymphocytes obtained from patients diagnosed with AF (n = 10) vs patients with no known arrhythmias (n = 11). DNA was then used for paired-end reduced representation bisulfite sequencing to quantify differentially methylated regions (DMRs). NetworkAnalyst tool was interrogated in order to build a specific protein-protein interaction network and identify hub differentially methylated genes (DMGs) in AF. We considered the centrality degree as main network topological measure and defined hubs those DMGs with a centrality degree > 10. Results The analysis provided 7,846 DMRs (P<0.05), associated with 5,373 annotated genes, among which 7,075 hyper-methylated and 771 hypo-methylated regions were uncovered (Figure 1A). Among the identified DMRs, 3,601 were located within gene promoters of which 3,513 were hypermethylated while 88 hypomethylated. Network analysis including the 50 top hyper- and hypomethylated DMGs, according to the differential methylation values, provided a heart specific subnetwork with 56 nodes and 611 edges and 51 seeds (Figure 1B). Although the DNA methylation trend showed a predominant hypermethylation pattern in CD4+ T cells from AF patients (Figure 1A), network analysis demonstrated that hypomethylation was prevalent in the top 15 hub DMGs. In particular, GSTK1, WWP1, PSMD6, TFRC, CDK5R1, HSPG2, WDFY3, WT1, USP49, ACOT7, LAMB2, ITGB7, SHARPIN, and PLXNB2 emerged as hypomethylated hub DMGs, while AIFM1, PFDN1, LMO3, CDK5RAP2, COL4A1, SEPT8, and CYTH4 resulted hypermethylated hub DMGs. Interestingly, several genes are involved in pathological mechanisms of AF in cardiac tissues such as inflammation, fibrosis and cardiac remodeling. Conclusions Our preliminary data represent the first global methylation analysis in circulating CD4+ T cells in AF. The validation of specific genomic regions and the detection of molecular pathways characterized by aberrant DNA methylation changes may help to clarify pathological mechanisms underlying AF, thus providing an important translational impact for novel diagnostic biomarker discovery and therapeutic target identification.Figure 1

Deubiquitylating Enzymes in Cancer and Immunity.

Deubiquitylating enzymes (DUBs) maintain relative homeostasis of the cellular ubiquitome by removing the post-translational modification ubiquitin moiety from substrates. Numerous DUBs have been demonstrated specificity for cleaving a certain type of ubiquitin linkage or positions within ubiquitin chains. Moreover, several DUBs perform functions through specific protein-protein interactions in a catalytically independent manner, which further expands the versatility and complexity of DUBs' functions. Dysregulation of DUBs disrupts the dynamic equilibrium of ubiquitome and causes various diseases, especially cancer and immune disorders. This review summarizes the Janus-faced roles of DUBs in cancer including proteasomal degradation, DNA repair, apoptosis, and tumor metastasis, as well as in immunity involving innate immune receptor signaling and inflammatory and autoimmune disorders. The prospects and challenges for the clinical development of DUB inhibitors are further discussed. The review provides a comprehensive understanding of the multi-faced roles of DUBs in cancer and immunity.

Accurate computational design of three-dimensional protein crystals.

Protein crystallization plays a central role in structural biology. Despite this, the process of crystallization remains poorly understood and highly empirical, with crystal contacts, lattice packing arrangements and space group preferences being largely unpredictable. Programming protein crystallization through precisely engineered side-chain-side-chain interactions across protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach for designing three-dimensional protein crystals with prespecified lattice architectures at atomic accuracy that hierarchically constrains the overall number of degrees of freedom of the system. We design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into >100 µm three-dimensional crystals. The structures of these crystals are nearly identical to the computational design models, closely corresponding in both overall architecture and the specific protein-protein interactions. The dimensions of the crystal unit cell can be systematically redesigned while retaining the space group symmetry and overall architecture, and the crystals are extremely porous and highly stable. Our approach enables the computational design of protein crystals with high accuracy, and the designed protein crystals, which have both structural and assembly information encoded in their primary sequences, provide a powerful platform for biological materials engineering.

Discovery of benzyloxy benzamide derivatives as potent neuroprotective agents against ischemic stroke

Aberrant activation of N-methyl-d-aspartate receptors (NMDAR) and the resulting neuronal nitric oxide synthase (nNOS) excessive activation play crucial pathogenic roles in neuronal damage caused by stroke. Disrupting postsynaptic density protein 95 (PSD95)-nNOS protein-protein interaction (PPI) has been proposed as a potential therapeutic strategy for ischemic stroke without incurring the unwanted side effects of direct NMDAR antagonism. Based on a specific PSD95-nNOS PPI inhibitor (SCR4026), we conducted a detailed study on structure-activity relationship (SAR) to discover a series of novel benzyloxy benzamide derivatives. Here, our efforts resulted in the best 29 (LY836) with improved neuroprotective activities in primary cortical neurons from glutamate-induced damage and drug-like properties. Whereafter, co-immunoprecipitation experiment demonstrated that 29 significantly blocked PSD95-nNOS association in cultured cortical neurons. Furthermore, 29 displayed good pharmacokinetic properties (T1/2 = 4.26 and 4.08 h after oral and intravenous administration, respectively) and exhibited powerful therapeutic effects in rats subjected to middle cerebral artery occlusion (MCAO) by reducing infarct size and neurological deficit score. These findings suggested that compound 29 may be a promising neuroprotection agent for the treatment of ischemic stroke.

A disordered region controls cBAF activity via condensation and partner recruitment

Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.

Gibbs Energy and Gene Expression Combined as a New Technique for Selecting Drug Targets for Inhibiting Specific Protein-Protein Interactions.

One of the most important aspects of successful cancer therapy is the identification of a target protein for inhibition interaction. Conventionally, this consists of screening a panel of genes to assess which is mutated and then developing a small molecule to inhibit the interaction of two proteins or to simply inhibit a specific protein from all interactions. In previous work, we have proposed computational methods that analyze protein-protein networks using both topological approaches and thermodynamic quantification provided by Gibbs free energy. In order to make these approaches both easier to implement and free of arbitrary topological filtration criteria, in the present paper, we propose a modification of the topological-thermodynamic analysis, which focuses on the selection of the most thermodynamically stable proteins and their subnetwork interaction partners with the highest expression levels. We illustrate the implementation of the new approach with two specific cases, glioblastoma (glioma brain tumors) and chronic lymphatic leukoma (CLL), based on the publicly available patient-derived datasets. We also discuss how this can be used in clinical practice in connection with the availability of approved and investigational drugs.

Predator: Predicting the impact of cancer somatic mutations on protein-protein interactions.

Since many biological processes are governed by protein-protein interactions, understanding which mutations lead to a disruption in these interactions is profoundly important for cancer research. Most of the existing methods focus on the stability of the protein without considering the specific effects of a mutation on its interactions with other proteins. Here, we focus on somatic mutations that appear on the interface regions of the protein and predict the interactions that would be affected by a mutation of interest. We build an ensemble model, Predator, that classifies the interface mutations as disruptive or nondisruptive based on the predicted effects of mutations on specific protein-protein interactions. We show that Predator outperforms existing approaches in literature in terms of prediction accuracy. We then apply Predator on various TCGA cancer cohorts and perform comprehensive analysis at cohort level, patient level, and gene level in determining the genes whose interface mutations tend to yield a disruption in its interactions. The predictions obtained by Predator shed light on interesting patterns on several genes for each cohort regarding their potential as cancer drivers. Our analyses further reveal that the identified genes and their frequently disrupted partners exhibit patterns of mutually exclusivity across cancer cohorts under study.

Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.