Biology subjects

Goldbaum, F. A.

Publications and source records attributed to Goldbaum, F. A..

4 recordsLinked to original sources

bioRxiv2024

Cryo-EM structures of engineered Shiga toxin-based immunogens capable of eliciting neutralizing antibodies with therapeutic potential against Hemolytic Uremic Syndrome

Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is a serious disease that causes renal failure predominantly in children. Despite its significant impact, there are currently no licensed vaccines or effective therapies available. The B subunits of Shiga toxins 1 and 2 (Stx1B and Stx2B) are suitable targets for developing neutralizing antibodies, but their pentameric assembly is unstable when isolated from the whole toxin. Taking advantage of the oligomeric symmetry shared between Stx1B and Stx2B with the lumazine synthase from Brucella spp. (BLS), we have previously engineered the chimeric toxoids BLS-Stx1B and BLS-Stx2B as immunogens to generate therapeutic equine polyclonal antibodies. The resulting product (INM004) has successfully passed phases 1 and 2 clinical trials, and a phase 3 has been launched in Argentina and seven European countries. In this work, we present the cryo-EM structures of BLS-Stx1B and BLS-Stx2B, which confirm that these engineered immunogens effectively stabilize the StxB pentamers. Moreover, our results reveal that both chimeric constructs present high flexibility at their extremes, corresponding to motions of the StxBs with respect to the BLS core. Additionally, we present structural evidence of the interaction between the chimeras and polyclonal Fab (pFab) fragments derived from INM004, demonstrating that the elicited neutralizing antibodies block most of the interaction surface of the toxins with their cellular receptors. These findings further validate this promising antibody-based therapy for mitigating STEC-HUS and demonstrate that the BLS-Stx1B and BLS-Stx2B chimeras are potential candidates for developing a human vaccine.

immunology↗

bioRxiv2022

A fijivirus major viroplasm protein shows RNA-stimulated ATPase activity by adopting pentameric and hexameric assemblies of dimers

The fijivirus Mal de Rio Cuarto virus (MRCV) causes a devastating maize disease. Its non-structural protein P9-1, which shows ATPase and RNA binding activities, is the major component of the intracellular viroplasm where virus replication takes place. Here, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures (VLS) in vivo and for the protein multimerization in vitro. Employing an integrative structural approach, we found that the C-arm is dispensable for P9-1 dimer assembly, but essential for the formation of doughnut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops oriented towards the inner pore of the structures, where RNA binding sites and conditional proteasome-mediated degradation signals (PEST) were predicted. In vitro assays demonstrated that ssRNA binding is favored towards P9-1 (do)decamers over the dimeric {Delta}C-arm version. In addition, although both P9-1 and P9-1{Delta}C-arm catalyze the hydrolysis of ATP with similar activity values, an RNA-stimulated ATPase activity was only observed in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. Computational studies revealed a stronger preference of phosphate moieties to the decamer in the pore and the C-arm regions, suggesting that the allosteric communication between the ATP and RNA binding sites is favored with this protein arrangement. Overall, our work reveals the structural versatility of a major viroplasm protein providing unprecedented insights into fijivirus viroplasm assembly and function and establishes the structural basis for the development of antiviral strategies against the Mal de Rio Cuarto crop disease.

biochemistry↗

bioRxiv2021

Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level

Light sensing allows organisms to adapt to constantly changing environmental factors. Phytochromes constitute a widespread biological photoreceptor family that typically interconvert between two photostates called Pr (red light-absorbing) and Pfr (far-red light-absorbing). Despite the vast structural information reported on phytochromes, the lack of full-length structures at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here we present three crystallographic structures from the plant pathogen Xanthomonas campestris virulence regulator bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structural findings, combined with mutational, biochemical and computational studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level, from the isomerization of the chromophore and the {beta}-sheet/-helix tongue transition to the remodeling of the quaternary assembly of the protein. TEASERCrystal structures of the full-length bacteriophytochrome XccBphP in both Pr and Pfr states unveil photoswitching mechanism

biophysics↗

bioRxiv2020

Crystal structures of peanut lectin in the presence of synthetic β-N- and β-S-galactosidesdisclose evidences for the recognition of different glycomimetic ligands

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation, and tumor metastasis. Hence, the structural studies of lectin-synthetic glycan complexes are essential for understanding the lectin recognition processes and the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular recognition process. Among them, peanut lectin (PNA) is highly relevant in the glycobiology field, because of its specificity for {beta}-galactosides, showing high affinity towards the Thomsen-Friedenreich (TF) antigen, a well-known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of {beta}-galactoside and {beta}-lactoside ligands. Herein, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable {beta}-N- and {beta}-S-galactosides. These complexes, along with computational simulations, disclosed key molecular binding interactions of the different sugars to PNA at the atomic level, revealing the role of specific water molecules in the protein-ligand recognition. Furthermore, binding affinity studies measured by isothermal titration calorimetry showed dissociation constant values in the micromolar range, as well as a positive glycoside cluster effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides qualitative structural rationale for the upcoming synthesis of optimized glycoclusters, designed for the study of lectin-mediated biological processes. The understanding of the recognition of {beta}-N- and {beta}-S-galactosides with PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands not belonging to the family of O-linked glycosides.

biochemistry↗