Structural basis of prostate-specific membrane antigen recognition by the A9g RNA aptamer.

Jakub Ptacek,Petr Kolenko,Petra Baranova,Paloma Giangrande,Zora Novakova,Shambhavi Shubham,Dong Zhang,Lucia Motlova,Sven Kruspe,Shi-Jie Chen,Cyril Barinka,Xiaoqin Zou,Liming Qiu,Barbora Havlinova

doi:10.1093/nar/gkaa494

Abstract

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.

Highlights

Prostate-specific membrane antigen (PSMA) is a membrane-tethered glycoprotein naturally expressed in various tissues, including the central nervous system, kidney, small intestine and liver [1,2,3,4,5,6,7]
PSMA is overexpressed on the surface of prostate cancer (PCa) cells compared to the limited expression levels observed in healthy prostate tissue, and this overexpression is significantly correlated to poor disease prognosis [8]
The extracellular part of human PSMA was co-crystallized with A9g and 2(phosphonomethyl)-pentanedioic acid (2-PMPA) at the 1:1.1:14 molar ratio. 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) is a reversible PSMA-specific inhibitor competing with the substrate in the active site of PSMA with the inhibition constant in a picomolar range. 2-PMPA was included in the crystallization droplets as our previous experiments revealed that the presence of the inhibitor greatly increases the probability of obtaining diffraction-quality crystals [63]

Summary

Introduction

Prostate-specific membrane antigen (PSMA) is a membrane-tethered glycoprotein naturally expressed in various tissues, including the central nervous system, kidney, small intestine and liver [1,2,3,4,5,6,7]. PSMA is overexpressed on the surface of prostate cancer (PCa) cells compared to the limited expression levels observed in healthy prostate tissue, and this overexpression is significantly correlated to poor disease prognosis [8]. Various approaches targeting PSMA have been developed since 1987 when PSMA was discovered as an antigen recognized by the 7E11-C5 monoclonal antibody (7E11-mAb) raised against LNCaP (a PSMApositive prostate adenocarcinoma-derived cell line) lysates [17]. A host of second-generation PSMA-specific antibodies have been prepared and engineered to small antibody fragments, humanized antibodies, radio-labeled derivatives, toxin fusions or bispecific

Methods

Results

Conclusion