A neutralizing antibody prevents postfusion transition of measles virus fusion protein.

Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.

The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.

Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.

Potent Omicron-neutralizing antibodies isolated from a patient vaccinated 6 months before Omicron emergence.

Therapeutic antibodies are an important tool in the arsenal against coronavirus infection. However, most antibodies developed early in the pandemic have lost most or all efficacy against newly emergent strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly those of the Omicron lineage. Here, we report the identification of a panel of vaccinee-derived antibodies that have broad-spectrum neutralization activity. Structural and biochemical characterization of the three broadest-spectrum antibodies reveal complementary footprints and differing requirements for avidity to overcome variant-associated mutations in their binding footprints. In the K18 mouse model of infection, these three antibodies exhibit protective efficacy against BA.1 and BA.2 infection. This study highlights the resilience and vulnerabilities of SARS-CoV-2 antibodies and provides road maps for further development of broad-spectrum therapeutics.

Structural basis for antibody-mediated neutralization of lymphocytic choriomeningitis virus.

The mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a globally distributed zoonotic pathogen that can be lethal in immunocompromised patients and can cause severe birth defects if acquired during pregnancy. The structure of the trimeric surface glycoprotein, essential for entry, vaccine design, and antibody neutralization, remains unknown. Here, we present the cryoelectron microscopy (cryo-EM) structure of the LCMV surface glycoprotein (GP) in its trimeric pre-fusion assembly both alone and in complex with a rationally engineered monoclonal neutralizing antibody termed 18.5C-M28 (M28). Additionally, we show that passive administration of M28, either as a prophylactic or therapeutic, protects mice from LCMV clone 13 (LCMVcl13) challenge. Our study illuminates not only the overall structural organization of LCMV GP and the mechanism for its inhibition by M28 but also presents a promising therapeutic candidate to prevent severe or fatal disease in individuals who are at risk of infection by a virus that poses a threat worldwide.

A metabolite binding protein moonlights as a bile-responsive chaperone.

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.

The cryo-EM structure of the human uromodulin filament core reveals a unique assembly mechanism.

The glycoprotein uromodulin (UMOD) is the most abundant protein in human urine and forms filamentous homopolymers that encapsulate and aggregate uropathogens, promoting pathogen clearance by urine excretion. Despite its critical role in the innate immune response against urinary tract infections, the structural basis and mechanism of UMOD polymerization remained unknown. Here, we present the cryo-EM structure of the UMOD filament core at 3.5 Å resolution, comprised of the bipartite zona pellucida (ZP) module in a helical arrangement with a rise of ~65 Å and a twist of ~180°. The immunoglobulin-like ZPN and ZPC subdomains of each monomer are separated by a long linker that interacts with the preceding ZPC and following ZPN subdomains by ß-sheet complementation. The unique filament architecture suggests an assembly mechanism in which subunit incorporation could be synchronized with proteolytic cleavage of the C-terminal pro-peptide that anchors assembly-incompetent UMOD precursors to the membrane.

Architecture and function of human uromodulin filaments in urinary tract infections.

Uromodulin is the most abundant protein in human urine, and it forms filaments that antagonize the adhesion of uropathogens; however, the filament structure and mechanism of protection remain poorly understood. We used cryo-electron tomography to show that the uromodulin filament consists of a zigzag-shaped backbone with laterally protruding arms. N-glycosylation mapping and biophysical assays revealed that uromodulin acts as a multivalent ligand for the bacterial type 1 pilus adhesin, presenting specific epitopes on the regularly spaced arms. Imaging of uromodulin-uropathogen interactions in vitro and in patient urine showed that uromodulin filaments associate with uropathogens and mediate bacterial aggregation, which likely prevents adhesion and allows clearance by micturition. These results provide a framework for understanding uromodulin in urinary tract infections and in its more enigmatic roles in physiology and disease.

Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.

Adhesive type 1 pili from enteroinvasive, Gram-negative bacteria mediate attachment to host cells. Up to 3000 copies of the main pilus subunit, FimA, assemble into the filamentous, helical quaternary structure of the pilus rod via a mechanism termed donor-strand complementation, in which the N-terminal extension of each subunit, the donor strand, is inserted into the incomplete immunoglobulin-like fold of the preceding FimA subunit. For FimA from Escherichia coli, it has been previously shown that the protein can also adopt a monomeric, self-complemented conformation in which the donor strand is inserted intramolecularly in the opposite orientation relative to that observed for FimA polymers. Notably, soluble FimA monomers can act as apoptosis inhibitors in epithelial cells after uptake of type 1-piliated pathogens. Here, we show that the FimA orthologues from Escherichia coli, Shigella flexneri, and Salmonella enterica can all fold to form self-complemented monomers. We solved X-ray structures of all three FimA monomers at 0.89-1.69 Å resolutions, revealing identical, intramolecular donor-strand complementation mechanisms. Our results also showed that the pseudo-palindromic sequences of the donor strands in all FimA proteins permit their alternative folding possibilities. All FimA monomers proved to be 50-60 kJ/mol less stable against unfolding than their pilus rod-like counterparts (which exhibited very high energy barriers of unfolding and refolding). We conclude that the ability of FimA to adopt an alternative, monomeric state with anti-apoptotic activity is a general feature of FimA proteins of type 1-piliated bacteria.