Summary CorA, the major Mg2+ uptake system in prokaryotes, is gated by intracellular Mg2+ (KD ~1–2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg2+-bound and Mg2+-free conditions, but EPR spectroscopic studies reveal large Mg2+-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg2+-bound “closed” conformation and in two “open” Mg2+-free states at resolutions of 3.8 A, 7.1 A and 7.1 A, respectively. In the absence of bound Mg2+, four of the five subunits are displaced to variable extents (~10 to ~25 A) by hinge-like motions at the stalk helix as large as ~35°. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg2+, open, asymmetric conformational states, is thus the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.

Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein–coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward β-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs.

SUMMARY Cells possess specialized machinery to direct the insertion of membrane proteins into the lipid bilayer. In bacteria, the essential protein YidC inserts certain proteins into the plasma membrane, and eukaryotic orthologs are present in the mitochondrial inner membrane and the chloroplast thylakoid membrane. The existence of homologous insertases in archaea has been proposed based on phylogenetic analysis. However, limited sequence identity, distinct architecture, and the absence of experimental data have made this assignment ambiguous. Here we describe the 3.5-Å crystal structure of an archaeal DUF106 protein from Methanocaldococcus jannaschii (Mj0480), revealing a lipid-exposed hydrophilic surface presented by a conserved YidC-like fold. Functional analysis reveals selective binding of Mj0480 to ribosomes displaying a stalled YidC substrate, and a direct interaction between the buried hydrophilic surface of Mj0480 and the nascent chain. These data provide direct experimental evidence that the archaeal DUF106 proteins are YidC/Oxa1/Alb3-like insertases of the archaeal plasma membrane.

SUMMARY A major challenge in membrane biophysics is to define the mechanistic linkages between a protein’s conformational transitions and its function. We describe a novel approach to stabilize transient functional states of membrane proteins in native-like lipid environments allowing for their structural and biochemical characterization. This is accomplished by combining the power of antibody Fab-based phage display selection with the benefits of embedding membrane protein targets in lipid-filled nanodiscs. In addition to providing a stabilizing lipid environment, nanodiscs afford significant technical advantages over detergent-based formats. This enables the production of a rich pool of high performance Fab binders that can be used as crystallization chaperones, as fiducial markers for single particle cryo-EM and as probes of different conformational states. Moreover, nanodisc generated Fabs can be used to identify detergents that best mimic native membrane environments for use in biophysical studies.

BackgroundT cell checkpoint immunotherapies have shown promising results in the clinic, but most patients remain non-responsive. CD47-signal regulatory protein alpha (SIRPα) myeloid checkpoint blockade has shown early clinical activity in hematologic malignancies. However, CD47 expression on peripheral blood limits αCD47 antibody selectivity and thus efficacy in solid tumors.MethodsTo improve the antibody selectivity and therapeutic window, we developed a novel affinity-tuned bispecific antibody targeting CD47 and programmed death-ligand 1 (PD-L1) to antagonize both innate and adaptive immune checkpoint pathways. This PD-L1-targeted CD47 bispecific antibody was designed with potent affinity for PD-L1 and moderate affinity for CD47 to achieve preferential binding on tumor and myeloid cells expressing PD-L1 in the tumor microenvironment (TME).ResultsThe antibody design reduced binding on red blood cells and enhanced selectivity to the TME, improving the therapeutic window compared with αCD47 and its combination with αPD-L1 in syngeneic tumor models. Mechanistically, both myeloid and T cells were activated and contributed to antitumor activity of αCD47/PD-L1 bispecific antibody. Distinct from αCD47 and αPD-L1 monotherapies or combination therapies, single-cell RNA sequencing (scRNA-seq) and gene expression analysis revealed that the bispecific treatment resulted in unique innate activation, including pattern recognition receptor-mediated induction of type I interferon pathways and antigen presentation in dendritic cells and macrophage populations. Furthermore, treatment increased the Tcf7+ stem-like progenitor CD8 T cell population in the TME and promoted its differentiation to an effector-like state. Consistent with mouse data, the compounds were well tolerated and demonstrated robust myeloid and T cell activation in non-human primates (NHPs). Notably, RNA-seq analysis in NHPs provided evidence that the innate activation was mainly contributed by CD47-SIRPα but not PD-L1-PD-1 blockade from the bispecific antibody.ConclusionThese findings provide novel mechanistic insights into how myeloid and T cells can be uniquely modulated by the dual innate and adaptive checkpoint antibody and demonstrate its potential in clinical development (NCT04881045) to improve patient outcomes over current PD-(L)1 and CD47-targeted therapies.

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Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones.

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca-binding site (Ca), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca to a cytoplasmic site (Ca). An X-ray structure with Ca removed and a near-atomic resolution cryo-EM structure with Ca removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca ions for activation.