Fatty acid transporter MFSD2A is a multifunctional gatekeeper in brain and placenta.

MFSD2A mediates uptake of the essential fatty acid DHA across the blood–brain barrier. Separately, via interactions with syncytin-2, MFSD2A contributes to the formation of the mother–fetus placental boundary. Cryo-EM analysis of a human MFSD2A–syncytin-2 complex provides new insights into how MFSD2A performs these dual roles.

Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance.

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively.

Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport.

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9.

Ribosomal protein L7/L12 is required for GTPase translation factors EF-G, RF3, and IF2 to bind in their GTP state to 70S ribosomes.

Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5' triphosphate hydrolase (GTPase) activity of elongation factor G (EF-G) requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from Escherichia coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2, and LepA.

The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood.

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes.

The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by 2 classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously was investigated by x-ray crystallography.

Characterization and solution structure of the factor VIII C2 domain in a ternary complex with classical and non-classical inhibitor antibodies.

The most significant complication for patients with severe cases of congenital or acquired hemophilia A is the development of inhibitor antibodies against coagulation factor VIII (fVIII). The C2 domain of fVIII is a significant antigenic target of anti-fVIII antibodies. Here, we have utilized small angle x-ray scattering (SAXS) and biochemical techniques to characterize interactions between two different classes of anti-C2 domain inhibitor antibodies and the isolated C2 domain.

Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4.

Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.

Expression, purification, and analysis of unknown translation factors from Escherichia coli: A synthesis approach.

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation.