Biochemical and Structural Insights into Carbonic Anhydrase XII/ Fab6A10 Complex Vincenzo Alterio 1, y , Markus Kellner 2, y , Davide Esposito 1 , Friederike Liesche-Starnecker 3 , Silvia Bua 4 , Claudiu T. Supuran 4 , Simona Maria Monti 1 , Reinhard Zeidler 2,5 and Giuseppina De Simone 1 1 - Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, Naples, 80134, Italy 2 - Department of Gene Vectors, Helmholtz Center for Environmental Health, Munich, Germany 3 - School of Medicine, Institute of Pathology, Department of Neuropathology, Technical University Munich, Munich, Germany 4 - Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universit aDegli Studi di Firenze, Via U. Schiff 6, Sesto Fiorentino, Florence, 50019, Italy 5 - Department of Otorhinolaryngology, Klinikum der Universit€ at München, Munich, Germany Correspondence to Giuseppina De Simone, Simona Maria Monti, and Reinhard Zeidler: Reinhard Zeidler is to be contacted at: Department of Gene Vectors, Helmholtz Center for Environmental Health, Munich, Germany. marmonti@unina.it, zeidler@helmholtz-muenchen.de, gdesimon@unina.it https://doi.org/10.1016/j.jmb.2019.10.022 Abstract 6A10 is a CA XII inhibitory monoclonal antibody, which was demonstrated to reduce the growth of cancer cells in vitro and in a xenograft model of lung cancer. It was also shown to enhance chemosensitivity of multiresistant cancer cell lines and to significantly reduce the number of lung metastases in combination with doxorubicin in mice carrying human triple-negative breast cancer xenografts. Starting from these data, we report here on the development of the 6A10 antigen-binding fragment (Fab), termed Fab6A10, and its functional, biochemical, and structural characterization. In vitro binding and inhibition assays demonstrated that Fab6A10 selectively binds and inhibits CA XII, whereas immunohistochemistry experiments highlighted its capability to stain malignant glioma cells in contrast to the surrounding brain tissue. Finally, the crystallographic structure of CA XII/Fab6A10 complex provided insights into the inhibition mechanism of Fab6A10, showing that upon binding, it obstructs the substrate access to the enzyme active site and interacts with CA XII His64 freezing it in its out conformation. Altogether, these data indicate Fab6A10 as a new promising therapeutic tool against cancer. © 2019 Elsevier Ltd. All rights reserved. Introduction Human carbonic anhydrases (hCAs) represent a family of enzymes that catalyze the reversible hydration of CO 2 to HCO 3  ions and protons [1,2]. There are 12 catalytically active isoforms known in humans (CAs IeIV, VA and VB, VI, VII, IX, XIIeXIV) that differ with respect to tissue and organ distribu- tion, as well as catalytic efficiency and cellular localization. Despite these differences, hCAs share a high level of three-dimensional similarity, a zinc ion in the active site, and the same two-step catalytic mechanism described by Eqs. (1) and (2) [1]: EZn 2þ  H 2 O þ B%EZn 2þ  OH  þ BH þ ð2Þ The first step of this mechanism involves the nucleophilic attack of a Zn 2þ -bound hydroxide on a CO 2 molecule bound in a hydrophobic pocket within EZn 2þ  OH  þ CO 2 %EZn 2þ  HCO  3 % H 2 O EZn 2þ  H 2 O þ HCO  3 ð1Þ YJMBI-66308; No. of pages: 12; 4C: Please cite this article as: V. Alterio, M. Kellner, D. Esposito, et al., Biochemical and Structural Insights into Carbonic Anhydrase XII/ Fab6A10 Complex, Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2019.10.022 0022-2836/© 2019 Elsevier Ltd. All rights reserved. Journal of Molecular Biology (xxxx) xx, xxx