Prostate-specific membrane antigen (PSMA) is a well-characterized tumor marker associated with prostate cancer and neovasculature of most solid tumors. PSMA-specific ligands are thus being developed to deliver imaging or therapeutic agents to cancer cells. Here, we report on a crystal structure of human PSMA in complex with A9g, a 43-bp PSMA-specific RNA aptamer, that was determined to the 2.2 Å resolution limit. The analysis of the PSMA/aptamer interface allows for identification of key interactions critical for nanomolar binding affinity and high selectivity of A9g for human PSMA. Combined with in silico modeling, site-directed mutagenesis, inhibition experiments and cell-based assays, the structure also provides an insight into structural changes of the aptamer and PSMA upon complex formation, mechanistic explanation for inhibition of the PSMA enzymatic activity by A9g as well as its ligand-selective competition with small molecules targeting the internal pocket of the enzyme. Additionally, comparison with published protein-RNA aptamer structures pointed toward more general features governing protein-aptamer interactions. Finally, our findings can be exploited for the structure-assisted design of future A9g-based derivatives with improved binding and stability characteristics.

Dalton Cardiovascular Research Center University of Missouri Columbia MO USA

Department of Internal Medicine University of Iowa Iowa City IA 52242 USA

Department of Physics and Astronomy University of Missouri Columbia MO USA

Department of Physics and Astronomy University of Missouri Columbia MO USA Dalton Cardiovascular Research Center University of Missouri Columbia MO USA Department of Biochemistry Institute for Data Science and Informatics University of Missouri Columbia MO USA

Department of Physics and Astronomy University of Missouri Columbia MO USA Department of Biochemistry Institute for Data Science and Informatics University of Missouri Columbia MO USA

Laboratory of Structural Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec 25250 Czech Republic

Laboratory of Structural Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec 25250 Czech Republic Faculty of Nuclear Sciences and Physical Engineering Czech Technical University Prague Brehova 7 Prague 11519 Czech Republic

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$a Ptacek, Jakub $u Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec 25250, Czech Republic.

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$a Structural basis of prostate-specific membrane antigen recognition by the A9g RNA aptamer / $c J. Ptacek, D. Zhang, L. Qiu, S. Kruspe, L. Motlova, P. Kolenko, Z. Novakova, S. Shubham, B. Havlinova, P. Baranova, SJ. Chen, X. Zou, P. Giangrande, C. Barinka,

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$a Prostate-specific membrane antigen (PSMA) is a well-characterized tumor marker associated with prostate cancer and neovasculature of most solid tumors. PSMA-specific ligands are thus being developed to deliver imaging or therapeutic agents to cancer cells. Here, we report on a crystal structure of human PSMA in complex with A9g, a 43-bp PSMA-specific RNA aptamer, that was determined to the 2.2 Å resolution limit. The analysis of the PSMA/aptamer interface allows for identification of key interactions critical for nanomolar binding affinity and high selectivity of A9g for human PSMA. Combined with in silico modeling, site-directed mutagenesis, inhibition experiments and cell-based assays, the structure also provides an insight into structural changes of the aptamer and PSMA upon complex formation, mechanistic explanation for inhibition of the PSMA enzymatic activity by A9g as well as its ligand-selective competition with small molecules targeting the internal pocket of the enzyme. Additionally, comparison with published protein-RNA aptamer structures pointed toward more general features governing protein-aptamer interactions. Finally, our findings can be exploited for the structure-assisted design of future A9g-based derivatives with improved binding and stability characteristics.

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$a Zhang, Dong $u Department of Physics and Astronomy, University of Missouri, Columbia, MO, USA.

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$a Qiu, Liming $u Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA.

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$a Kruspe, Sven $u Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

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$a Motlova, Lucia $u Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec 25250, Czech Republic.

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$a Kolenko, Petr $u Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec 25250, Czech Republic. Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Brehova 7, Prague 11519, Czech Republic.

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$a Novakova, Zora $u Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec 25250, Czech Republic.

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$a Shubham, Shambhavi $u Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

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$a Havlinova, Barbora $u Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec 25250, Czech Republic.

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$a Baranova, Petra $u Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec 25250, Czech Republic.

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$a Chen, Shi-Jie $u Department of Physics and Astronomy, University of Missouri, Columbia, MO, USA. Department of Biochemistry, Institute for Data Science and Informatics, University of Missouri, Columbia, MO, USA.

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$a Zou, Xiaoqin $u Department of Physics and Astronomy, University of Missouri, Columbia, MO, USA. Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA. Department of Biochemistry, Institute for Data Science and Informatics, University of Missouri, Columbia, MO, USA.

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$a Giangrande, Paloma $u Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

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$a Barinka, Cyril $u Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec 25250, Czech Republic.

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