Junctions between the ER and plasma membrane (PM) are implicated in calcium homeostasis, non-vesicular lipid transfer, and other cellular functions. Two ER proteins that function both as tethers to the PM via a polybasic C-terminus motif and as phospholipid transporters are brain-enriched TMEM24 (C2CD2L) and its paralog C2CD2. We report that both proteins also form a complex with band 4.1 family members, which in turn bind PM proteins including cell adhesion molecules such as SynCAM 1. This complex enriches TMEM24 and C2CD2 containing ER/PM junctions at sites of cell contacts. Dynamic properties of TMEM24-dependent ER/PM junctions are impacted when band 4.1 is part of the junction, as TMEM24 at cell-adjacent ER/PM junctions is not shed from the PM by calcium rise, unlike TMEM24 at non-cell adjacent junctions. Lipid transport between the ER and the PM by TMEM24 and C2CD2 at sites where cells, including neurons, contact other cells may participate in adaptive responses to cell contact-dependent signaling.

Complementary structural biology approaches reveal how an agonist and a covalent inhibitor simultaneously bind to a nuclear receptor.

Copper chaperones of the ATX1 family are found in a wide range of organisms where these essential soluble carriers strictly control the transport of monovalent copper across the cytoplasm to various targets in diverse cellular compartments thereby preventing detrimental radical formation catalyzed by the free metal ion. Notably, the ATX1 family in plants contains two distinct forms of the cellular copper carrier. In addition to ATX1 having orthologs in other species, they also contain the copper chaperone CCH. The latter features an extra C-terminal extension whose function is still unknown. The secondary structure of this extension was predicted to be disordered in previous studies, although this has not been experimentally confirmed. Solution NMR studies on purified CCH presented in this study disclose that this region is intrinsically disordered regardless of the chaperone\'s copper loading state. Further biophysical analyses of the purified metallochaperone provide evidence that the C-terminal extension stabilizes chaperone dimerization in the copper-free and copper-bound states. A variant of CCH lacking the C-terminal extension, termed CCHΔ, shows weaker dimerization but similar copper binding. Computational studies further corroborate the stabilizing role of the C-terminal extension in chaperone dimerization and identify key residues that are vital to maintaining dimer stability.

Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a \"negative cooperativity\" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.

Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.

DSS1, essential for BRCA2-RAD51 dependent homologous recombination (HR), associates with the helical domain (HD) and OB fold 1 (OB1) of the BRCA2 DSS1/DNA-binding domain (DBD) which is frequently targeted by cancer-associated pathogenic variants. Herein, we reveal robust ss/dsDNA binding abilities in HD-OB1 subdomains and find that DSS1 shuts down HD-OB1\'s DNA binding to enable ssDNA targeting of the BRCA2-RAD51 complex. We show that C-terminal helix mutations of DSS1, including the cancer-associated R57Q mutation, disrupt this DSS1 regulation and permit dsDNA binding of HD-OB1/BRCA2-DBD. Importantly, these DSS1 mutations impair BRCA2/RAD51 ssDNA loading and focus formation and cause decreased HR efficiency, destabilization of stalled forks and R-loop accumulation, and hypersensitize cells to DNA-damaging agents. We propose that DSS1 restrains the intrinsic dsDNA binding of BRCA2-DBD to ensure BRCA2/RAD51 targeting to ssDNA, thereby promoting optimal execution of HR, and potentially replication fork protection and R-loop suppression.

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1β. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.

Kisspeptin receptor (KISS1R), belonging to the class A peptide-GPCR family, plays a key role in the regulation of reproductive physiology after stimulation by kisspeptin and is regarded as an attractive drug target for reproductive diseases. Here, we demonstrated that KISS1R can couple to the Gi/o pathway besides the well-known Gq/11 pathway. We further resolved the cryo-electron microscopy (cryo-EM) structure of KISS1R-Gq and KISS1R-Gi complexes bound to the synthetic agonist TAK448 and structure of KISS1R-Gq complex bound to the endogenous agonist KP54. The high-resolution structures provided clear insights into mechanism of KISS1R recognition by its ligand and can facilitate the design of targeted drugs with high affinity to improve treatment effects. Moreover, the structural and functional analyses indicated that conformational differences in the extracellular loops (ECLs), intracellular loops (ICLs) of the receptor, and the \"wavy hook\" of the Gα subunit may account for the specificity of G protein coupling for KISS1R signaling.

Enhancing virtual screening enrichment has become an urgent problem in computational chemistry, driven by increasingly large databases of commercially available compounds, without a commensurate drop in in vitro screening costs. Docking these large databases is possible with cloud-scale computing. However, rapid docking necessitates compromises in scoring, often leading to poor enrichment and an abundance of false positives in docking results. This work describes a new scoring function composed of two parts - a knowledge-based component that predicts the probability of a particular atom type being in a particular receptor environment, and a tunable weight matrix that converts the probability predictions into a dimensionless score suitable for virtual screening enrichment. This score, the FitScore, represents the compatibility between the ligand and the binding site and is capable of a high degree of enrichment across standardized docking test sets.