Neuro-endocrine prostate cancer (NEPC) accounts for about 20% of lethal metastatic castration-resistant prostate cancer (CRPC). NEPC has the most aggressive biologic behavior of all prostate cancers and is associated with poor patient outcome. Effective treatment for NEPC is not available because NEPC exhibit distinct cell-surface expression profiles compared to other types of prostate cancer. Recently, the carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) (known as CEA or CD66e) was suggested to be a specific surface protein marker for NEPC. Therefore, we identified a new, fully-human anti-CEACAM5 monoclonal antibody, 1G9, which bound to the most proximal membrane domains, A3 and B3, of CEACAM5 with high affinity and specificity. It shows no off-target binding to other CEACAM family members, membrane distal domains of CEACAM5, or 5800 human membrane proteins. IgG1 1G9 exhibited CEACAM5-specific ADCC activity toward CEACAM5-positive prostate cancer cells in vitro and in vivo. Chimeric antigen receptor T cells (CAR-T) based on scFv 1G9 induced specific and strong antitumor activity in a mouse model of prostate cancer. Our results suggest that IgG1 and CAR-T cells based on 1G9 are promising candidate therapeutics for CEACAM5-positive NEPC and other cancers.

Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs "Duraleukin" and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs.

Catalysis and translocation of multi-subunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near atomic resolution and precise arrangement of key active site components have been elusive. Here we present the free electron laser (FEL) structure of a matched ATP-bound Pol II, revealing the full active site interaction network at the highest resolution to date, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structure indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/bridge helix (BH) interactions induce conformational changes that could propel translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the hyperactive Rpb1 T834P bridge helix mutant reveals rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10-100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of the dependence of crystal quality on crystal size.

The PTH receptor is to our knowledge one of the first G protein-coupled receptor (GPCR) found to sustain cAMP signaling after internalization of the ligand-receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned off.

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec-dGTPase, including an X-ray free-electron laser structure of the free Ec-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec-dGTPase residues at the end of the substrate-binding pocket mimic Watson-Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally "opening" a tyrosine gate allowing enhanced substrate binding.

The nucleocapsid (N) protein is one of the four structural proteins of the SARS-CoV-2 virus and plays a crucial role in viral genome organization and, hence, replication and pathogenicity. The N-terminal domain (NNTD) binds to the genomic RNA and thus comprises a potential target for inhibitor and vaccine development. We determined the atomic-resolution structure of crystalline NNTD by integrating solid-state magic angle spinning (MAS) NMR and X-ray diffraction. Our combined approach provides atomic details of protein packing interfaces as well as information about flexible regions as the N- and C-termini and the functionally important RNA binding, β-hairpin loop. In addition, ultrafast (100 kHz) MAS 1H-detected experiments permitted the assignment of side-chain proton chemical shifts not available by other means. The present structure offers guidance for designing therapeutic interventions against the SARS-CoV-2 infection.