A Q-transform-based deep learning model for the classification of atrial fibrillation types.

According to the World Health Organization (WHO), Atrial Fibrillation (AF) is emerging as a global epidemic, which has resulted in a need for techniques to accurately diagnose AF and its various subtypes. While the classification of cardiac arrhythmias with AF is common, distinguishing between AF subtypes is not. Accurate classification of AF subtypes is important for making better clinical decisions and for timely management of the disease. AI techniques are increasingly being considered for image classification and detection in various ailments, as they have shown promising results in improving diagnosis and treatment outcomes. This paper reports the development of a custom 2D Convolutional Neural Network (CNN) model with six layers to automatically differentiate Non-Atrial Fibrillation (Non-AF) rhythm from Paroxysmal Atrial Fibrillation (PAF) and Persistent Atrial Fibrillation (PsAF) rhythms from ECG images. ECG signals were obtained from a publicly available database and segmented into 10-second segments. Applying Constant Q-Transform (CQT) to the segmented ECG signals created a time-frequency depiction, yielding 98,966 images for Non-AF, 16,497 images for PAF, and 52,861 images for PsAF. Due to class imbalance in the PAF and PsAF classes, data augmentation techniques were utilized to increase the number of PAF and PsAF images to match the count of Non-AF images. The training, validation, and testing ratios were 0.7, 0.15, and 0.15, respectively. The training set consisted of 207,828 images, whereas the testing and validation set consisted of 44,538 images and 44,532 images, respectively. The proposed model achieved accuracy, precision, sensitivity, specificity, and F1 score values of 0.98, 0.98, 0.98, 0.97, and 0.98, respectively. This model has the potential to assist physicians in selecting personalized AF treatment and reducing misdiagnosis.

Sensing for survival: specialised regulatory mechanisms of Type III secretion systems in Gram-negative pathogens.

For centuries, Gram-negative pathogens have infected the human population and been responsible for numerous diseases in animals and plants. Despite advancements in therapeutics, Gram-negative pathogens continue to evolve, with some having developed multi-drug resistant phenotypes. For the successful control of infections caused by these bacteria, we need to widen our understanding of the mechanisms of host-pathogen interactions. Gram-negative pathogens utilise an array of effector proteins to hijack the host system to survive within the host environment. These proteins are secreted into the host system via various secretion systems, including the integral Type III secretion system (T3SS). The T3SS spans two bacterial membranes and one host membrane to deliver effector proteins (virulence factors) into the host cell. This multifaceted process has multiple layers of regulation and various checkpoints. In this review, we highlight the multiple strategies adopted by these pathogens to regulate or maintain virulence via the T3SS, encompassing the regulation of small molecules to sense and communicate with the host system, as well as master regulators, gatekeepers, chaperones, and other effectors that recognise successful host contact. Further, we discuss the regulatory links between the T3SS and other systems, like flagella and metabolic pathways including the tricarboxylic acid (TCA) cycle, anaerobic metabolism, and stringent cell response.

Recognition of Aedes aegypti Mosquito Saliva Protein LTRIN by the Human Receptor LTßR for Controlling the Immune Response.

Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTßR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTßR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTßR using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTßR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV.

Structural and functional characterization of Aedes aegypti pupal cuticle protein that controls dengue virus infection.

The pupal cuticle protein from Aedes aegypti (AaPC) inhibits dengue virus (DENV) infection; however, the underlying mechanism of this inhibition remains unknown. Here, we report that AaPC is an intrinsically disordered protein and interacts with domain I/II of the DENV envelope protein via residues Asp59, Asp61, Glu71, Asp73, Ser75, and Asp80. AaPC can directly bind to and cause the aggregation of DENV, which in turn blocks virus infection during the virus-cell fusion stage. AaPC may also influence viral recognition and attachment by interacting with human immune receptors DC-SIGN and CD4. These findings enhance our understanding of the role of AaPC in mitigating viral infection and suggest that AaPC is a potential target for developing inhibitors or antibodies to control dengue virus infection.

Bacterioferritin nanocage structures uncover the biomineralization process in ferritins.

Iron is an essential element involved in various metabolic processes. The ferritin family of proteins forms nanocage assembly and is involved in iron oxidation, storage, and mineralization. Although several structures of human ferritins and bacterioferritins have been solved, there is still no complete structure that shows both the trapped Fe-biomineral cluster and the nanocage. Furthermore, whereas the mechanism of iron trafficking has been explained using various approaches, structural details on the biomineralization process (i.e. the formation of the mineral itself) are generally lacking. Here, we report the cryo-electron microscopy (cryo-EM) structures of apoform and biomineral bound form (holoforms) of the Streptomyces coelicolor bacterioferritin (ScBfr) nanocage and the subunit crystal structure. The holoforms show different stages of Fe-biomineral accumulation inside the nanocage, in which the connections exist in two of the fourfold channels of the nanocage between the C-terminal of the ScBfr monomers and the Fe-biomineral cluster. The mutation and truncation of the bacterioferritin residues involved in these connections significantly reduced the iron and phosphate binding in comparison with those of the wild type and together explain the underlying mechanism. Collectively, our results represent a prototype for the bacterioferritin nanocage, which reveals insight into its biomineralization and the potential channel for bacterioferritin-associated iron trafficking.

Structural Basis for the Enzymatic Activity of the HACE1 HECT-Type E3 Ligase Through N-Terminal Helix Dimerization.

HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo-EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N-terminal AKR domain, a middle (MID) domain, and a C-terminal HECT domain. Its unique G-shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N-terminal helix of one monomer restricts access to the C-terminal domain of the other. The in vitro ubiquitination assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.

CatBoost-based improved detection of P-wave changes in sinus rhythm and tachycardia conditions: a lead selection study.

Examining P-wave morphological changes in Electrocardiogram (ECG) is essential for characterizing atrial arrhythmias. However, standard 12-lead ECGsuffer from diagnostic redundancy due to low signal-to-noise ratio of P-waves. To address this issue, various optimal leads have been proposed for improved atrial activity recording, but the right selection among these leads is crucial for enhancing diagnostic efficacy. This study proposes an automated lead selection technique using the CatBoost machine learning (ML) model to improve the detection of P-wave changes among optimal bipolar leads under different heart rates. ECGs were obtained from healthy participants with a mean age of 25 ± 3.81 years (34% women), including 114 in sinus rhythm (SR) and 38 in sinus tachycardia (ST). The recordings were made using a newly designed atrial lead system (ALS), standard limb lead (SLL), modified limb lead (MLL), modified Lewis lead (LLM) and P-lead. P-wave features and Atrioventricular (AV) ratio were extracted for statistical analysis and ML classification. The optimum ML model was chosen to identify the best-performing optimal lead, which was selected based on the SLL metrics among different ML classifiers. CatBoost was found to outperform the other ML models in SLL-II with the highest accuracy and sensitivity of 0.82 and 0.90, respectively. The CatBoost model, amid other optimal leads, gave the best results for AL-I and AL-II (0.86 and 0.83 in accuracy and 0.91 and 0.93 in sensitivity). The developed CatBoost model selected AL-I and AL-II as the top two best-performing optimal leads for the enhanced acquisition of P-wave changes, which may be useful for diagnosing atrial arrhythmias.

Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.

Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

A novel allosteric site employs a conserved inhibition mechanism in human kidney-type glutaminase.

Glutaminase catalyses the metabolic process called glutaminolysis. Cancer cells harness glutaminolysis to increase energy reserves under stressful conditions for rapid proliferation. Glutaminases are upregulated in many tumours. In humans, the kidney-type glutaminase (KGA) isoform is highly expressed in the kidney, brain, intestine, foetal liver, lymphocytes and in many tumours. Glutaminase inhibition is shown to be effective in controlling cancers. Previously, we and others reported the inhibition mechanism of KGA using various inhibitors that target the active and allosteric sites of the enzyme. Here, we report the identification of a novel allosteric site in KGA using the compound DDP through its complex crystal structure combined with mutational and hydrogen-deuterium exchange mass spectrometry studies. This allosteric site is located at the dimer interface, situated ~ 31 Å away from the previously identified allosteric site and ~ 32 Å away from the active site. Remarkably, the mechanism of inhibition is conserved, irrespective of which allosteric pocket is targeted, causing the same conformational changes in the key loop near the active site (Glu312-Pro329) and subsequent enzyme inactivation. Contrary to the previously identified allosteric site, the identified new allosteric site is primarily hydrophilic. This site could be effectively targeted for the synthesis of specific and potent water-soluble inhibitors of glutaminase, which will lead to the development of anticancer drugs.

Identification of Aedes aegypti salivary gland proteins interacting with human immune receptor proteins.

Mosquito saliva proteins modulate the human immune and hemostatic systems and control mosquito-borne pathogenic infections. One mechanism through which mosquito proteins may influence host immunity and hemostasis is their interactions with key human receptor proteins that may act as receptors for or coordinate attacks against invading pathogens. Here, using pull-down assays and proteomics-based mass spectrometry, we identified 11 Ae. aegypti salivary gland proteins (SGPs) (e.g., apyrase, Ae. aegypti venom allergen-1 [AaVA-1], neutrophil stimulating protein 1 [NeSt1], and D7 proteins), that interact with one or more of five human receptor proteins (cluster of differentiation 4 [CD4], CD14, CD86, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin [DC-SIGN], and Toll-like receptor 4 [TLR4]). We focused on CD4- and DC-SIGN-interacting proteins and confirmed that CD4 directly interacts with AaVA-1, D7, and NeST1 recombinant proteins and that AaVA-1 showed a moderate interaction with DC-SIGN using ELISA. Bacteria responsive protein 1 (AgBR1), an Ae. aegypti saliva protein reported to enhance ZIKV infection in humans but that was not identified in our pull-down assay moderately interacts with CD4 in the ELISA assay. Functionally, we showed that AaVA-1 and NeST1 proteins promoted activation of CD4+ T cells. We propose the possible impact of these interactions and effects on mosquito-borne viral infections such as dengue, Zika, and chikungunya viruses. Overall, this study provides key insight into the vector-host (protein-protein) interaction network and suggests roles for these interactions in mosquito-borne viral infections.

Scaffold stability and P14' residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4.

Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most-if not all-of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only three residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14' position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.

Mapping of molecular interactions between human E3 ligase TRIM69 and Dengue virus NS3 protease using hydrogen-deuterium exchange mass spectrometry.

Tripartite motif (TRIM) E3 ligases target specific substrates, including viral proteins, for proteasomal degradation, and are thus essential regulators of the innate antiviral response. TRIM69 ubiquitinates the non-structural NS3 protein of Dengue virus for its degradation by the host machinery. This antiviral strategy abrogates the immunosuppression mediated by the NS2B-NS3 protease complex. To understand how this host-driven antiviral response against Dengue virus, we sought to define the mode of interaction between human TRIM69 and Dengue NS2B-NS3 and the subsequent polyubiquitination of the protease by the E3 ligase. We show that NS2B-NS3Δpro is sufficient as a substrate for ubiquitination by TRIM69 using ELISA and in vitro assays. Using hydrogen-deuterium exchange mass spectrometry (HDXMS), we mapped the interface of the interaction between TRIM69 and NS2B-NS3Δpro, and propose a rationale for the binding and subsequent ubiquitination process. Furthermore, through sequence analysis, we showed that the regions targeted by TRIM69 on the DENV-2 NS3 protease (NS3Δpro) are well conserved across DENV serotypes and other flaviviruses, including Zika virus, West Nile virus, and Japanese encephalitis virus. Our results show the direct interactions of TRIM69 with viral proteins, provide mechanistic insights at a molecular level, and highlight the functional relevance of TRIM69 interacting with the Dengue viral protein. Collectively, our findings suggest that TRIM69 may act as a pan-antiflaviviral restriction factor.

Identification of putative binding interface of PI(3,5)P2 lipid on rice black-streaked dwarf virus (RBSDV) P10 protein.

Rice black-streaked dwarf virus (RBSDV) is an important reovirus that infects both plants and its transmission vector small brown planthopper, causing severe crop loss. High affinity binding between RBSDV P10 and PI(3,5)P2 lipid layer was measured using biolayer interferometry (BLI). Subcellular co-localization of PI(3,5)P2 and RBSDV P10 was observed on membranous structures in insect cells with stochastic optical reconstruction microscopy (STORM) imaging. Putative interacting sites of PI(3,5)P2 lipid on a computational predicted RBSDV P10 structure were mapped to its "C-domain" (250-470 aa), using HDXMS data. The BLI and STORM results showed binding and co-localization of RBSDV P10, and PI(3,5)P2 on vesicle-like membranous structures were corroborated with the prediction of the binding interface. Understanding the lipid binding sites on viral proteins will lead to developing strategies to block viral-lipid interaction and disrupt viral pathogenesis in insect vectors and to block virus transmission and achieve disease control of crops in the field.

Sequence preference and scaffolding requirement for the inhibition of human neutrophil elastase by ecotin peptide.

Human neutrophil elastase (hNE) is an abundant serine protease that is a major constituent of lung elastolytic activity. However, when secreted in excess, if not properly attenuated by selective inhibitor proteins, it can have detrimental effects on host tissues, leading to chronic lung inflammation and non-small cell lung cancer. To improve upon the design of inhibitors against hNE for therapeutic applications, here, we report the crystal structure of hNE in complex with an ecotin (ET)-derived peptide inhibitor. We show that the peptide binds in the nonprime substrate binding site. Unexpectedly, compared with full-length (FL) ET, we find that our short linear peptides and circular amide backbone-linked peptides of ET are incapable of efficient hNE inhibition. Our structural insights point to a preferred amino acid sequence and the potential benefit of a scaffold for optimal binding and function of the peptide inhibitor, both of which are retained in the FL ET protein. These findings will aid in the development of effective peptide-based inhibitors against hNE for targeted therapy.

Bacterial antagonism of Chromobacterium haemolyticum and characterization of its putative type VI secretion system.

This study reports the isolation of a new Chromobacterium haemolyticum strain named WI5 from a hydroponic farming facility. WI5 exhibited remarkable bacterial antagonistic properties, eliminating Salmonella, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus (initial inoculum load ∼105 CFU/ml) in dual-species co-culture biofilms. Antagonism was strictly contact-dependent and highly influenced by nutrient availability. Next, we identified a complete suite of putative Type VI secretion system (T6SS) genes in the WI5 genome, annotated the gene locus architecture, and determined the crystal structure of hallmark T6SS tube protein Hcp1, which revealed a hexameric ring structure with an outer and inner diameter of 77 and 45 Å, respectively. Structural comparison with homologs showed differences in the key loops connecting the ß-strands in which the conserved residues are located, suggesting a role of these residues in the protein function. The T6SS is well-known to facilitate interbacterial competition, and the putative T6SS characterized herein might be responsible for the remarkable antagonism by C. haemolyticum WI5. Collectively, these findings shed light on the nature of bacterial antagonism and a putative key virulence determinant of C. haemolyticum, which might aid in further understanding its potential ecological role in natural habitats.

Structure of Aedes aegypti procarboxypeptidase B1 and its binding with Dengue virus for controlling infection.

Metallocarboxypeptidases play critical roles in the development of mosquitoes and influence pathogen/parasite infection of the mosquito midgut. Here, we report the crystal structure of Aedes aegypti procarboxypeptidase B1 (PCPBAe1), characterized its substrate specificity and mechanism of binding to and inhibiting Dengue virus (DENV). We show that the activated PCPBAe1 (CPBAe1) hydrolyzes both Arg- and Lys-substrates, which is modulated by residues Asp251 and Ser239 Notably, these residues are conserved in CPBs across mosquito species, possibly required for efficient digestion of basic dietary residues that are necessary for mosquito reproduction and development. Importantly, we characterized the interaction between PCPBAe1 and DENV envelope (E) protein, virus-like particles, and infectious virions. We identified residues Asp18A, Glu19A, Glu85, Arg87, and Arg89 of PCPBAe1 are essential for interaction with DENV. PCPBAe1 maps to the dimeric interface of the E protein domains I/II (Lys64-Glu84, Val238-Val252, and Leu278-Leu287). Overall, our studies provide general insights into how the substrate-binding property of mosquito carboxypeptidases could be targeted to potentially control mosquito populations or proposes a mechanism by which PCPBAe1 binds to and inhibits DENV.

Crystal structure of Aedes aegypti trypsin inhibitor in complex with µ-plasmin reveals role for scaffold stability in Kazal-type serine protease inhibitor.

Kazal-type protease inhibitor specificity is believed to be determined by sequence of the reactive-site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with µ-plasmin, and compared its reactivities with other Kazal-type inhibitors, infestin-1 and infestin-4. We show that the shortened 99-loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive-site loop of infestin-4. In contrast, AaTI and infestin-1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys-V to Cys-VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys-V to Cys-VI region between AaTI and infestin-4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin-4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal-type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50.

Binding specificity of type three secretion system effector NleH2 to multi-cargo chaperone CesT and their phosphorylation.

Gram-negative pathogens like Enteropathogenic Escherichia coli (EPEC) utilize the type three secretion system (T3SS) to translocate various effector proteins that are needed to "hijack" the host system for pathogenic survival. Specialized T3SS chaperones inside bacterial cells stabilize these effector proteins and facilitate their translocation. CesT is a unique multi-cargo chaperone that interacts with and translocates ~10 different effector proteins. Here, we report the specific interaction between CesT and its key effector, NleH2, and explore the potential role of NleH2 as a kinase for CesT phosphorylation. First, we identified the chaperone-binding domain (CBD; 19-97aa) of NleH2, and mapped the specific interaction sites for both CesT and NleH2. The N- and C-terminal residues of the CBD interact with the dimeric interface of CesT. Further, we compared the CesT binding to NleH2, to that of another key effector Tir and with the global carbon regulator CsrA. Notably, the effectors have the binding regions at the ß-sheet core and dimer interface of CesT, whereas the CsrA regulator interacts predominantly through the C-terminal region, which is found ~17 Å away from the effectors-binding sites. Next, we showed that NleH2 remains an active kinase even as a complex with CesT and is responsible for its autophosphorylation as well as phosphorylation of CesT at Tyr153. Collectively, our findings enhance the understanding of the role of multi-cargo chaperone CesT in orchestrating effector translocation through T3SS.

Structure of Aedes aegypti carboxypeptidase B1-inhibitor complex uncover the disparity between mosquito and non-mosquito insect carboxypeptidase inhibition mechanism.

Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI; MW 4 kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and ß8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect Helicoverpa zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki  = 14.7 nM) is marginally less than that of bovine CPA1 (Ki  = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy.

Structural basis for p50RhoGAP BCH domain-mediated regulation of Rho inactivation.

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the ß5-strand of the BCH domain is involved in an intermolecular ß-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the ß5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain-containing proteins.