Kaituo Wang

Zinc is essential for life yet toxic in excess, rendering homeostatic control vital for all organisms. Transition metal transporting P-type (P _IB -) ATPase proteins are critical for this balance but remain poorly understood at the molecular level. Here, we present the inward-open structure of a dimeric zinc-transporting P _IB -type ATPase, resolved at 3.6 Å resolution. Biochemical analyses and molecular dynamics simulations suggest that the N-terminal tail, harboring two so-called metal binding domains (MBDs), auto-inhibits the ATPase core and serves as a metal-sensitive sensor, releasing inhibition when intracellular metal levels rise. Contrary to the prevailing uptake models, the structure reveals an electronegatively charged uptake region that directly exposes a metal-binding cysteine of the invariant CPC-motif. We propose that a histidine-rich segment, positioned after the MBD closest to the core, donates ions to the CPC site. Consequently, an intricate interplay between the regulatory and ion-providing N-terminus and the ATPase core is present, safeguarding against zinc toxicity. Collectively, our findings shed light on the regulation and transport mechanism of P _IB -ATPases, with implications for fundamental biology and the potential to inform future biotechnological and translational applications.

Endoplasmic reticulum (ER) membrane resident P5A-ATPases broadly affect protein biogenesis and quality control, and yet their molecular function remains debated. Here, we report cryo-EM structures of a P5A-ATPase, CtSpf1, covering multiple transport intermediates of the E1 → E1-ATP → E1P-ADP → E1P → E2P → E2.P_i → E2 → E1 cycle. In the E2P and E2.P_i states a cleft spans the entire membrane, holding a polypeptide cargo molecule. The cargo includes an ER luminal extension, pinpointed as the C-terminus in the E2.P_i state, which reenters the membrane in E2P. The E1 structure harbors a cytosol-facing cavity that is blocked by an insertion we refer to as the Plug-domain. The Plug-domain is nestled to key ATPase features and is displaced in the E1P-ADP and E1P states. Collectively, our findings are compatible with a broad range of proteins as cargo, with the P5A-ATPases serving a role in membrane removal of helices, although insertion/secretion cannot be excluded, as well as with a mechanistic role of the Plug-domain.

The continuous emergence of SARS-CoV-2 variants of concern has rendered many therapeutic monoclonal antibodies (mAbs) ineffective. To date, there are no clinically authorized therapeutic antibodies effective against the recently circulating Omicron sub-lineages BA.2.86 and JN.1. Here, we report the isolation of broad and potent neutralizing human mAbs (HuMabs) from a healthcare worker infected with SARS-CoV-2 early in the pandemic. These include a genetically unique HuMab, named K501SP6, which can neutralize different Omicron sub-lineages, including BQ.1, XBB.1, BA.2.86 and JN.1, by targeting a highly conserved epitope on the N terminal domain, as well as an RBD-specific HuMab (K501SP3) with high potency towards earlier circulating variants that was escaped by the more recent Omicron sub-lineages through spike F486 and E484 substitutions. Characterizing SARS-CoV-2 spike-specific HuMabs, including broadly reactive non-RBD-specific HuMabs, can give insight into the immune mechanisms involved in neutralization and immune evasion, which can be a valuable addition to already existing SARS-CoV-2 therapies.

Fifty-eight million individuals worldwide are affected by chronic hepatitis C virus (HCV) infection, a primary driver of liver cancer for which no vaccine is available¹. The HCV envelope proteins E1 and E2 form a heterodimer (E1/E2), which is the target for neutralizing antibodies². However, the higher-order organization of these E1/E2 heterodimers, as well as that of any Hepacivirus envelope protein complex, remains unknown. Here we determined the cryo-electron microscopy structure of two E1/E2 heterodimers in a homodimeric arrangement. We reveal how the homodimer is established at the molecular level and provide insights into neutralizing antibody evasion and membrane fusion by HCV, as orchestrated by E2 motifs such as hypervariable region 1 and antigenic site 412, as well as the organization of the transmembrane helices, including two internal to E1. This study addresses long-standing questions on the higher-order oligomeric arrangement of Hepacivirus envelope proteins and provides a critical framework in the design of novel HCV vaccine antigens.

Molecular similarities between embryonic and malignant cells can be exploited to target tumors through specific signatures absent in healthy adult tissues. One such embryonic signature tumors express is oncofetal chondroitin sulfate (ofCS), which supports disease progression and dissemination in cancer. Here, we report the identification and characterization of phage display-derived antibody fragments recognizing two distinct ofCS epitopes. These antibody fragments show binding affinity to ofCS in the low nanomolar range across a broad selection of solid tumor types in vitro and in vivo with minimal binding to normal, inflamed, or benign tumor tissues. Anti-ofCS antibody drug conjugates and bispecific immune cell engagers based on these targeting moieties disrupt tumor progression in animal models of human and murine cancers. Thus, anti-ofCS antibody fragments hold promise for the development of broadly effective therapeutic and diagnostic applications targeting human malignancies.

Copper transporting P-type (P_1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P_1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P_1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD⁻¹, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD⁻¹, MBD⁻², likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P_1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P_1B-1-disorders and ongoing clinical trials.

Biogenic monoamines, vital transmitters orchestrating neurological, endocrinal, and immunological functions^1-5, are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release^6,7. Harnessing proton antiport, VMATs enrich monoamines ~10,000-fold and sequester neurotoxicants to protect neurons^8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension, and drug addiction^1,8,11-16. However, the structural mechanisms underlying these actions remain elusive. Here, we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines, the Parkinsonism-inducing MPP⁺, the psychostimulant amphetamine, and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, while other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favored transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket possessing polar sites for specificity and a wrist-and-fist shape for versatility. Variations of this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop novel therapeutics for neurodegenerative diseases and substance abuse.

Worldwide, 58 million individuals suffer from chronic hepatitis C virus (HCV) infection, a primary driver of liver cancer. The HCV envelope proteins, E1 and E2, form a heterodimer, which is the target for neutralizing antibodies. Despite high-resolution structural models of partial heterodimer elements, the structural landscape of higher-order E1/E2 oligomers remains unexplored. We determined a ~3.5 Å cryo-electron microscopy structure of membrane-extracted HCV E1/E2 in a homodimeric arrangement. This structure includes detailed information on the homodimer interface, the E2-binding pocket for hypervariable region 1, antigenic site 412 conformation, and the organization of the E1/E2 transmembrane regions, including one internal to E1. This higher-order E1/E2 assembly could play a pivotal role in the design of novel vaccine antigens better mimicking E1/E2 complexes on the HCV particle.

The continuous emergence of SARS-CoV-2 variants of concern with mutated spike receptor binding domains has rendered many therapeutic mAbs ineffective. To date, there are no clinically authorized therapeutic antibodies effective against the predominant circulating sub-lineages BQ and XBB. Here, we report the isolation of broad and potent neutralizing HuMabs from a Danish healthcare worker infected with SARS-CoV-2 early in the pandemic. These HuMabs include a novel and genetically unique non-RBD-specific HuMab (K501SP6) which can neutralize Omicron sub-lineages BQ and XBB, and an RBD-specific HuMab (K501SP3) with high potency towards earlier circulating variants but was escaped by Omicron sub-lineages BA.5, BQ and XBB through F486 and E484 substitutions. Characterizing SARS-CoV-2 spike-specific HuMabs, including broadly reactive non-RBD-specific HuMabs, can give insight into the immune mechanisms involved in neutralization and immune evasion, which can be a valuable addition to already existing SARS-CoV-2 therapies.

Ex vivo structure determination of macromolecules from native source is gaining increasing attention from the scientific community, as the method can be employed to dissect the function of important, multi-component molecular machines. However, the existing ex vivo procedures often require genome manipulation or availability high-affinity binders, limiting the general applicability. Here, we report simple yet robust principles for isolation of protein complexes from enriched native biological material, enabling cryoEM-facilitated high-resolution structure determination. We report the structures of ten separate membrane and soluble protein complexes determined from spinach leaves. Moreover, the developed pipeline is likely adaptable to essentially any biological system. As such, the approach may represent an attractive avenue for future structural proteomics efforts.