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Human calcium sensing receptor (CaSR) senses calcium ion concentrations in vivo and is an important class of drug targets. Mutations in the receptor can lead to disorders of calcium homeostasis, including hypercalcemia and hypocalcemia. Here, 127 CaSR-targeted nanobodies were generated from camels, and four nanobodies with inhibitory function were further identified. Among these nanobodies, NB32 can effectively inhibit the mobilization of intracellular calcium ions (Ca2+ i) and suppress the G 12/13 and ERK 1/2 signaling pathways downstream of CaSR. Moreover, it enhanced the inhibitory effect of the calcilytics as a negative allosteric modulator (NAM). We determined the structure of complex and found NB32 bound to LB2 (Ligand-binding 2) domain of CaSR to prevent the interaction of LB2 domains of two protomers to stabilize the inactive state of CaSR. • 127 CaSR-targeting nanobodies were screened from camels. • NB32 effectively inhibit the mobilization of intracellular calcium ions. • NB32 enhanced the inhibitory effect of the calcilytics as a negative allosteric modulator (NAM). • Cryo-EM revealed that NB32 binds to the LB2 domain of CaSR. [ABSTRACT FROM AUTHOR]

Metal-organic frameworks (MOFs) are promising adsorbents for legacy per-/polyfluoroalkyl substances (PFASs), but they are being replaced by emerging PFASs. The effects of varying carbon chains and functional groups of emerging PFASs on their adsorption behavior on MOFs require attention. This study systematically revealed the structure-adsorption relationships and interaction mechanisms of legacy and emerging PFASs on a typical MOF MIL-101(Cr). It also presented an approach reflecting the average electronegativity of PFAS moieties for adsorption prediction. We demonstrated that short-chain or sulfonate PFASs showed higher adsorption capacities (μmol/g) on MIL-101(Cr) than their long-chain or carboxylate counterparts, respectively. Compared with linear PFASs, their branched isomers were found to exhibit a higher adsorption potential on MIL-101(Cr). In addition, the introduction of ether bond into PFAS molecule (e.g., hexafluoropropylene oxide dimeric acid, GenX) increased the adsorption capacity, while the replacement of CF 2 moieties in PFAS molecule with CH 2 moieties (e.g., 6:2 fluorotelomer sulfonate, 6:2 FTS) caused a decrease in adsorption. Divalent ions (such as Ca2+ and SO 4 2−) and solution pH have a greater effect on the adsorption of PFASs containing ether bonds or more CF 2 moieties. PFAS adsorption on MIL-101(Cr) was governed by electrostatic interaction, complexation, hydrogen bonding, π-CF interaction, and π-anion interaction as well as steric effects, which were associated with the molecular electronegativity and chain length of each PFAS. The average electronegativity of individual moieties (named M e) for each PFAS was estimated and found to show a significantly positive correlation with the corresponding adsorption capacity on MIL-101(Cr). The removal rates of major PFASs in contaminated groundwater by MIL-101(Cr) were also correlated with the corresponding M e values. These findings will assist with the adsorption prediction for a wide range of PFASs and contribute to tailoring efficient MOF materials. [Display omitted] • Short-chain, branched, or sulfonate PFAS show a higher adsorption on MIL-101(Cr). • In PFAS, ether bond aids its adsorption on MIL-101(Cr), but H/F exchanges weaken it. • Divalent ions and pH have a greater effect on more electronegative PFAS adsorption. • Electrostatic, complex, π-CF interactions, and steric effects govern PFAS adsorption. • Moiety electronegativity of each PFAS can be used for adsorption prediction on MOFs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Crystal Structure of Two Non-competitive Nanobodies and SARS-CoV-2 RBD. • Two nanobodies neutralize SARS-CoV-2 including omicron variants by targeting ultra-conservative epitopes. • Developed a multivalent nanobody with high affinity and competitiveness. The evolving SARS-CoV-2 Omicron strain has repeatedly caused widespread disease epidemics, and effective antibody drugs continue to be in short supply. Here, we identified a batch of nanobodies with high affinity for receptor binding domain (RBD) of SARS-CoV-2 spike protein, separated them into three classes using high performance liquid chromatography (HPLC), and then resolved the crystal structure of the ternary complexes of two non-competing nanobodies (NB1C6 and NB1B5) with RBD using X-ray crystallography. The structures showed that NB1B5 and NB1C6 bind to the left and right flank of the RBD, respectively, and that the binding epitopes are highly conserved cryptic sites in all SARS-CoV-2 mutant strains, as well as that NB1B5 can effectively block the ACE2. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, and have a high affinity and neutralization potency for omicron, potentially inhibiting viral escape. The binding sites of these two nanobodies are relatively conserved, which help guide the structural design of antibodies targeting future variants of SARS-CoV-2 to combat COVID-19 epidemics and pandemics. [ABSTRACT FROM AUTHOR]

As one of the receptors of the TAM family, AXL plays a vital role in stem cell maintenance, angiogenesis, immune escape of viruses and drug resistance against tumors. In this study, the truncated extracellular segment containing two immunoglobulin-like domains of human AXL (AXL-IG), which has been confirmed to bind growth arrest specific 6 (GAS6) by structural studies [1], was expressed in a prokaryotic expression system and then purified. Immunizing camelid with the purified AXL-IG as antigen could lead to the production of unique nanobodies composed of only variable domain of heavy chain of heavy-chain antibody (VHH), which are around 15 kD and stable. We screened out a nanobody A-LY01 specific binding to AXL-IG. We further determined the affinity of A-LY01 to AXL-IG and revealed that A-LY01 could specifically recognize full-length AXL on the surface of HEK 293T/17 cells. Our study provides appropriate support for the development of diagnostic reagents and antibody therapeutics targeting AXL. • AXL-IG protein was expressed in E. coli WK6. • A nanobody phage library was constructed. • A nanobody A-LY01 specifically binds to AXL was screened out by phage display technology. [ABSTRACT FROM AUTHOR]