Nearly half of the ~5,000 human membrane proteins need to assemble into stoichiometric complexes as part of their biogenesis at the endoplasmic reticulum (ER) membrane. How ER-resident biogenesis factors coordinate membrane insertion, folding and assembly is not well understood. Here we demonstrate that the human ER membrane protein complex (EMC) insertase additionally acts as a chaperone to facilitate the assembly of heterotrimeric voltage-gated calcium channels (Cav). Using function-separating mutations and inhibitory nanobodies, we show that nascent Cav channels are degraded prematurely when EMC's chaperone function is selectively perturbed. Blocking EMC's chaperone function impaired Cav-dependent cardiomyocyte contraction. EMC engagement of the pore-forming Cav α-subunit occurred cotranslationally and required the Cav α-subunit's first transmembrane domain bundle to protrude from the nascent ribosome-multipass translocon complex. Our findings establish a chaperone function for the EMC and suggest that biogenesis of multibundle membrane proteins may require a highly orchestrated, cotranslational interplay among ER biogenesis factors.

The cell surface proteome governs processes essential for development, immunity and disease, yet its large-scale spatial organization remains uncharted. Here, we systematically map the surface proteome of human T lymphocytes and B lymphoblasts using proximity labeling of 85 antigens. Coupled with data-independent acquisition mass spectrometry, this approach reveals functional modules, including known complexes such as the T cell receptor alongside clusters not previously implicated in cell membrane functions. Notably, we identify mitochondrial proteins at the surface, suggesting that they may bear underappreciated roles at the plasma membrane. A machine learning classifier predicts surface protein associations, revealing the IL-10 receptor beta subunit's regulation of type I interferon signaling. Spatial modeling distinguishes widely dispersed from localized antigens, indicating active mechanisms of surface organization. In summary, this work provides a large-scale map of the human surfaceome and a resource for exploring its spatial and functional organization.

R2 retroelements are non-long-terminal-repeat retrotransposons that primarily insert into 28S ribosomal DNA loci across diverse eukaryotes. R2 elements home to their target sites through target-primed reverse transcription (TPRT), in which the R2 protein recognizes and nicks the target DNA to prime reverse transcription using an R2-encoding RNA as the template. The avian R2 system from Taeniopygia guttata (R2Tg) can insert a payload sequence within an RNA template into 28S ribosomal DNA loci in human cells. Here, we present cryo-electron microscopy structures of the R2Tg-template RNA-target DNA complex in the TPRT initiation state, both alone and bound to a Cas9-guide RNA complex. These structures provide mechanistic insights into R2Tg-mediated TPRT in its natural context and in the programmable gene integration system STITCHR (site-specific target-primed insertion through targeted CRISPR homing of retroelements). Furthermore, we combine these structural insights with computational protein design methods to engineer an enhanced STITCHR system.

Insights into the dynamics of G-protein-coupled receptors (GPCRs) remain limited. Here we present an integrative strategy that bridges computational and experimental biophysical methods for probing protein dynamics. To illustrate its potential, we developed GPCRmd-NMR, a platform that synergistically combines molecular dynamics (MD) simulations with high-resolution nuclear magnetic resonance (NMR) spectroscopy across the three-dimensional GPCRome. The resulting dataset enables structure-resolved analysis by applying the strategy across multiple GPCRs. We provide access to underexplored functional GPCR dynamics, receptor states and sites, including microswitches, hidden ligand entrance gateways and intrinsically disordered regions. Our integrated framework offers a solution to challenges faced by both experimental and computational researchers including the MD-NMR timescale gap.

Phage infection places strong selective pressure on bacteria to mount a rapid immune response to ensure survival. Although recent studies have uncovered a wide diversity of bacterial immune systems, how these systems function under the tight time constraints of phage infection remains poorly understood. Here we summarize structural, biochemical and genetic insights into bacterial immunity, focusing on steps that link infection sensing to activation and effector function. We highlight how conformational change, protein assembly and spatial organization shape immune activation, and how phages employ counter-defense strategies that disrupt these processes.

Norrin binds Frizzled 4 (FZD4), tetraspanin 12 (TSPAN12) and low-density lipoprotein receptor-related protein 5 (LRP5) to regulate angiogenesis and the integrity of the blood-brain barrier and inner blood-retina barrier (BRB). Aberrant Norrin signal causes retinopathies, BRB defects and deafness. Therapeutic mimetics of Norrin are currently in clinical trials for eye diseases. To characterize the mechanism of Norrin signal biogenesis, we determined the structure of a Norrin-FZD4-TSPAN12-LRP5 signaling complex using a de novo designed FZD4 binder as a 'protein glue'. Our structure shows that two curved Norrin homodimers bridge two FZD4-TSPAN12 heterodimers. LRP5LA3 simultaneously interacts with TSPAN12 and two Norrin homodimers, positioning the cytosolic FZD4 and LRP5 tails for signal activation. On the basis of our findings, we designed FZD4CRD-LRP5LA3 bispecific binders that act as Norrin surrogates. Our signaling assays showed that oligomerization of the Norrin surrogate enhances signaling potency. Thus, this work reveals how the Norrin signal is generated and may assist in the development of Norrin/Wnt mimetics.

DNA compaction by human condensin II is crucial for the correct organization of mitotic chromosomes. Mitotic activation of condensin II is a multilayered process, controlled mainly by the opposing activity of the MCPH1 and M18BP1 proteins, acting as an interphase inhibitor and a mitotic activator, respectively. Mitotic phosphorylation favors M18BP1 binding to condensin II, resulting in effective and timely chromosome compaction. However, the molecular mechanisms underlying condensin II activation in driving DNA compaction remain uncharacterized. Here, combining cryo-electron microscopy with single-molecule imaging, we unravel the mechanism of condensin II activation by M18BP1 and elucidate their role in DNA compaction. The specific interaction with phosphorylated M18BP1 induces conformational changes in the condensin II complex, relieving an autoinhibitory conformation and allowing stable DNA binding and DNA compaction. Beyond the activity of isolated condensin II complexes on DNA, we observe the emergence of multimeric species that withstand the forces applied by optical tweezers, suggesting that condensin II multimerization might contribute to the mechanical reinforcement of mitotic chromosomes.

Alternative splicing provides a pervasive means to expand proteome complexity, yet how it reorganizes protein interactions and cellular localization remains unclear. Here, we show that tissue-regulated longer exons preferably reshape local interaction patterns, whereas microexons fine-tune key interfaces linking distinct cellular processes. Integration of subcellular localization data further indicates that such rewiring can redistribute proteins within cells. By using a high-content imaging approach to systematically evaluate the influence of individual exons on protein localization, we show that 38% of the tested isoform pairs alter localization, with microexons, albeit typically shorter than five amino acids, accounting for a substantial proportion of these effects. Bioinformatic and structural analyses demonstrate that microexons can extend secondary structural regions and reposition charged residues, suggesting a potential to modulate local electrostatic environments. Consistent with this, biochemical analysis of a 4-aa microexon in sorting nexin 2, identified in our screen, confirms that residue insertion rather than side-chain chemistry drives key differences in protein localization through repositioning of a flanking, charged residue. Together, these findings suggest that tissue-regulated alternative splicing fine-tunes interface architecture, thus regulating protein assembly, localization and proteome organization.

Activation of heterotrimeric G proteins by G-protein-coupled receptors (GPCRs) requires large-scale opening of the Gα α-helical domain (AHD) to expose the nucleotide-binding site and facilitate GDP-GTP exchange. Although orthosteric ligands are known to modulate GPCR conformation, how these effects propagate to the bound G protein remains unclear. Using single-molecule Förster resonance energy transfer imaging, we monitored AHD motions in Gi proteins coupled to the μ-opioid receptor across different ligand- and nucleotide-bound states. We find that receptor ligands differentially modulate these dynamics from a binding site over 70 Å away, with higher-efficacy agonists accelerating transitions to an open, low-nucleotide-affinity conformation. These data also capture transient AHD intermediates during nucleotide exchange and suggest that allosteric control over the G protein conformational states provides an additional mechanism for encoding opioid efficacy and signaling outcomes.

Most eukaryotic membrane proteins are inserted into the membrane at the endoplasmic reticulum (ER). This essential process relies on molecular quality control machineries to prevent mistargeting and incorrect structure formation. Here, we show that the ER membrane protein complex (EMC) forms an evolutionarily conserved supercomplex with the P5A-ATPase Spf1/ATP13A1. Biochemical, structural and cellular analyses demonstrate that the EMC and Spf1 associate in a stoichiometric manner and retain their individual catalytic activities, the transmembrane domain (TMD) insertase function of the EMC and the TMD dislocase activity of Spf1. Our cryo-electron microscopy structures show that the EMC insertase cavity and the Spf1 dislocase cleft are positioned in close proximity within the membrane and reveal that the ATPase cycle of Spf1 remodels the interface between the two machineries and alters cytoplasmic accessibility of both substrate-binding cavities. Together, our study provides structural insights into how membrane protein insertion and extraction machineries can be spatially coordinated at the ER membrane, suggesting that functionally distinct proteostasis factors can assemble into hubs to support membrane protein biogenesis and quality control.