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1. Plasma membrane association and resistosome formation of plant helper immune receptors

   https://doi.org/10.1073/pnas.2222036120  

   Wang, Zaiqing; Liu, Xiaoxiao; Yu, Jie; Yin, Shuining; Cai, Wenjuan; Kim, Nak Hyun; El_Kasmi, Farid; Dangl, Jeffery L; Wan, Li (August 2023, Proceedings of the National Academy of Sciences)

   Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation. 

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2. Competition between Nucleic Acids and Intrinsically Disordered Regions within Proteins

   https://doi.org/10.1021/acs.accounts.5c00261  

   Wang, Xi; Levy, Yaakov; Iwahara, Junji (July 2025, Accounts of Chemical Research)

   Intrinsically disordered regions (IDRs) are important components of protein functionality, with their charge distribution serving as a key factor in determining their roles. Notably, many proteins possess IDRs that are highly negatively charged, characterized by sequences rich in aspartate (D) or glutamate (E) residues. Bioinformatic analyses indicate that negatively charged low-complexity IDRs are significantly more common than their positively charged counterparts rich in arginine (R) or lysine (K). For instance, sequences of 10 or more consecutive negatively charged residues (D or E) are present in 268 human proteins. In contrast, corresponding sequences of 10 or more consecutive positively charged residues (K or R) are present in only 12 human proteins. Interestingly, about 50% of proteins containing D/E tracts function as DNA-binding or RNA-binding proteins. Negatively charged IDRs can electrostatically mimic nucleic acids and dynamically compete with them for the DNA-binding domains (DBDs) or RNA-binding domains (RBDs) that are positively charged. This leads to a phenomenon known as autoinhibition, in which the negatively charged IDRs inhibit binding to nucleic acids by occupying the binding interfaces within the proteins through intramolecular interactions. Rather than merely reducing binding activity, negatively charged IDRs offer significant advantages for the functions of DNA/RNA-binding proteins. The dynamic competition between negatively charged IDRs and nucleic acids can accelerate the target search processes for these proteins. When a protein encounters DNA or RNA, the electrostatic repulsion force between the nucleic acids and the negatively charged IDRs can trigger conformational changes that allow the nucleic acids to access DBDs or RBDs. Additionally, when proteins are trapped at high-affinity non-target sites on DNA or RNA ("decoys"), the electrostatic repulsion from the negatively charged IDRs can rescue the proteins from these traps. Negatively charged IDRs act as gatekeepers, rejecting nonspecific ligands while allowing the target to access the molecular interfaces of DBDs or RBDs, which increases binding specificity. These IDRs can also promote proper protein folding, facilitate chromatin remodeling by displacing other proteins bound to DNA, and influence phase separation, affecting local pH. The functions of negatively charged IDRs can be regulated through protein-protein interactions, post-translational modifications, and proteolytic processing. These characteristics can be harnessed as tools for protein engineering. Some frame-shift mutations that convert negatively charged IDRs into positively charged ones are linked to human diseases. Therefore, it is crucial to understand the physicochemical properties and functional roles of negatively charged IDRs that compete with nucleic acids. 

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3. From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway

   https://doi.org/10.1186/s12860-023-00492-3  

   Zhao, Andy; Varady, Sophia; O’Kelley-Bangsberg, Madelyn; Deng, Vicki; Platenkamp, Amy; Wijngaard, Petra; Bern, Miriam; Gormley, Wyatt; Kushkowski, Elaine; Thompson, Kat; et al (October 2023, BMC Molecular and Cell Biology)

   Abstract The morphogenetic process of apical constriction, which relies on non-muscle myosin II (NMII) generated constriction of apical domains of epithelial cells, is key to the development of complex cellular patterns. Apical constriction occurs in almost all multicellular organisms, but one of the most well-characterized systems is the Folded-gastrulation (Fog)-induced apical constriction that occurs inDrosophila. The binding of Fog to its cognizant receptors Mist/Smog results in a signaling cascade that leads to the activation of NMII-generated contractility. Despite our knowledge of key molecular players involved in Fog signaling, we sought to explore whether other proteins have an undiscovered role in its regulation. We developed a computational method to predict unidentified candidate NMII regulators using a network of pairwise protein–protein interactions called an interactome. We first constructed aDrosophilainteractome of over 500,000 protein–protein interactions from several databases that curate high-throughput experiments. Next, we implemented several graph-based algorithms that predicted 14 proteins potentially involved in Fog signaling. To test these candidates, we used RNAi depletion in combination with a cellular contractility assay inDrosophilaS2R + cells, which respond to Fog by contracting in a stereotypical manner. Of the candidates we screened using this assay, two proteins, the serine/threonine phosphatase Flapwing and the putative guanylate kinase CG11811 were demonstrated to inhibit cellular contractility when depleted, suggestive of their roles as novel regulators of the Fog pathway. 

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4. How a highly acidic SH3 domain folds in the absence of its charged peptide target

   https://doi.org/10.1002/pro.4635  

   Jaramillo‐Martinez, Valeria; Dominguez, Matthew J.; Bell, Gemma M.; Souness, Megan E.; Carhart, Anna H.; Cuibus, M. Adriana; Masoumzadeh, Elahe; Lantz, Benjamin J.; Adkins, Aaron J.; Latham, Michael P.; et al (April 2023, Protein Science)

   Abstract Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye–Huckel screening and a nonspecific territorial ion‐binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short‐range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years. 

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5. Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel

   https://doi.org/10.1038/s41598-024-61274-1  

   Masse, Meranda_M; Guzman-Luna, Valeria; Varela, Angela_E; Mahfuza_Shapla, Ummay; Hutchinson, Rachel_B; Srivastava, Aniruddha; Wei, Wanting; Fuchs, Andrew_M; Cavagnero, Silvia (May 2024, Scientific Reports)

   Abstract In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp_1–140interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63–94 residues. Longer RNCs (apoHmp_1–189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface. 

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