Heim, J. B. | bioRxiv Science

bioRxiv2025

Structural and functional characterization of integrin a5-targeting antibodies for anti-angiogenic therapy

Integrins are heterodimeric receptors important for cell adhesion and signaling. Integrin 5{beta}1 is a key mediator of angiogenesis and its dysregulation is associated with tumor progression and metastasis. Despite numerous efforts, 5{beta}1-targeting therapeutics have been unsuccessful due to poor efficacy and off-target effects. A contributing factor is our limited understanding of how integrin conformation influences interactions with therapeutics. Using cell-based functional assays, patient-derived xenografts, biophysics, and electron microscopy, we shed light on these relationships by characterizing two anti-5{beta}1 antibodies, BIIG2 and MINT1526A. We show that both antibodies bind 5{beta}1 with nanomolar affinity, reduce tube formation in vitro, and bind overlapping epitopes that block fibronectin binding. However, using electron microscopy, we reveal that while BIIG2 binding does not substantially alter the conformational states, MINT1526A preferentially recognizes the bent conformation and restricts the conformational ensemble. These insights can guide which aspects to prioritize to improve the design of future integrin-targeted therapeutics.

biochemistry↗

bioRxiv2018

Crystal structures reveal that Lewis-x and fucose bind to secondary cholera toxin binding site - in contrast to fucosyl-GM1

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor - the cholera toxin (CT). Recent studies suggest that the GM1 receptor may not be the only target of the CT, and that fucosylated receptors such as Lewisx (Lex) and histo-blood group antigens may also be important for cellular uptake and toxicity. However, where and how Lex binds to the CT remains unclear. Here we report the high-resolution crystal structure (1.5 [A]) of the receptor-binding B-subunit of the CT bound to the Lex trisaccharide, and present matching SPR data for CT holotoxins. Lex, and also O_SCPCAPLC_SCPCAP-fucose alone (at 500-fold molar excess), bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. The two binding sites are likely connected by allosteric cross-talk, potentially affecting toxin uptake. We also discuss why secretors are protected from severe cholera.\n\nAuthor summaryCholera is a severe diarrhoeal disease that is still a major killer in many parts of the world, especially in regions struck by natural disasters and wars. However, some individuals experience milder cholera symptoms. These so-called secretors, who have blood group antigens also in their bodily fluids like their saliva and the slimy mucus layer covering their stomach and intestines, appear to be somewhat protected. Here we present detailed atomic structures of cholera toxin and quantitative binding data that give clues of the protective mechanisms. Interactions of the protein toxin with sugar molecules are of crucial importance both for toxicity and protection. In addition, we identify a new tool for biochemical studies, and lay the groundwork for the design of cholera drugs and vaccines that may save countless human lives.

biochemistry↗