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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 Lb2 Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2 Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2 Cas12a assumes a unique, elongated conformation, whereas the Lb2 Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2 Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2 Cas12a 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 Lb2 Cas12a 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.

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.

Obtaining pure and homogeneous protein samples is vital for protein biology studies, yet optimizing protein expression and purification methods can be time‐consuming because of variations in factors like expression conditions, buffer components, and fusion tags. With over 81 000 Protein Data Bank (PDB)‐associated articles as of October 2024, manual extraction of relevant methods is impractical. To streamline this process, an automated tool is developed by incorporating a large language model (LLM) to extract and classify key data from these articles. The information extraction accuracy is enhanced by a 2‐step‐LLM and a 3‐step‐prompt. The key findings include: 1) Tris buffer is used in 49.2% of cases, followed by 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) and phosphate buffers. 2) Polyhistidine tags dominate at 82.5%, followed by glutathione S‐transferase (GST) and maltose‐binding protein (MBP) tags. 3) E. coli expression is done at 16–20 °C, with induction period favoring 12–16 h (69.0%) over 3–6 h (14.3%). The statistical analyses highlight the correlation between protein properties and purification strategies. This tool is validated through two case studies: method bias for membrane protein purification, and crosslinker/detergent preferences for Cryo‐Electron Microscopy sample preparation. These findings provide a valuable resource for designing protein expression and purification experiments. An automated tool incorporating a large language model (LLM) streamlines the extraction and classification of protein expression and purification methods from 64 909 PDB‐associated articles. By identifying trends in buffer components, fusion tags, expression conditions, and crosslinker/detergent preferences, the tool provides valuable insights into the correlation between protein properties and purification strategies.

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA's active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

Nanobodies (Nbs) show promise in combating arboviruses. Their small size and stability enable blood–brain barrier (BBB) penetration, cryptic epitope binding, and reduced antibody-dependent enhancement (ADE). This forum highlights Nb advantages, challenges, and innovative delivery methods, emphasizing their transformative potential in arbovirus therapeutics and diagnostics. Nanobodies (Nbs) show promise in combating arboviruses. Their small size and stability enable blood–brain barrier (BBB) penetration, cryptic epitope binding, and reduced antibody-dependent enhancement (ADE). This forum highlights Nb advantages, challenges, and innovative delivery methods, emphasizing their transformative potential in arbovirus therapeutics and diagnostics.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein–protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP ( Y BCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the β5 strand. Our previous WT Y BCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant Y BCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in H BCH) as a conserved residue in the crucial β5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N 2 ) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions.

Hepatitis E virus （HEV）, a non-enveloped, positive-sense, single-stranded RNA virus, is a major cause of enteric hepatitis. Classified into the family Hepeviridae, HEV comprises four genotypes （genotypes 1-4）, which belong to a single serotype. We describe a monoclonal antibody （mAb）, 8G12, which equally recognizes all four genotypes of HEV, with -2.53-3.45 nM binding affinity. The mAb 8G12 has a protective, neutralizing capacity, which can significantly block virus infection in host cells. Animal studies with genotypes 1, 3 and 4 confirmed the cross-genotype neutralizing capacity of 8G12 and its effective prevention of hepatitis E disease. The complex crystal structures of 8G12 with the HEV E2s domain （the most protruded region of the virus capsid） of the abundant genotypes ! and 4 were determined at 4.0 and 2.3 ~, resolution, respectively. These structures revealed that 8G12 recognizes both gen- otypes through the epitopes in the E2s dimerization region. Structure-based mutagenesis and cell-model assays with virus-like particles identified several conserved residues （Glu549, Lys554 and Gly591） that are essential for 8G12 neutralization. Moreover, the epitope of 8G12 is identified as a key epitope involved in virus-host interactions. These findings will help develop a common strategy for the prevention of the most abundant form of HEV infection.

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.

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV-host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.