Communication between thrombin exosites: structural effects of the simultaneous binding of two bimodular aptamers

Human α-thrombin is a trypsin-like serine protease playing a pivotal role in the haemostatic pathway. Apart from the active site, thrombin has two electropositive patches on its surface, named exosites I and II, which determine the biological properties of the enzyme through the binding of substrates and cofactors [1,2]. The two exosites also represent the anchoring sites for aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity, by adopting defined and stable 3D structures [3,4]. New generation anti-thrombin aptamers, which adopt a mixed duplex/quadruplex structure and possess improved binding or anticoagulant properties, are currently under study [5]. Among them, NU172, the only thrombin binding aptamer in advanced clinical trial, recognizes exosite I with its quadruplex domain [6]. On the other hand, HD22_27mer oligonucleotide binds exosite II with both quadruplex and duplex domains [7]. Both these bimodular aptamers show high affinity toward their target. Recently, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on both thrombin exosites [8-11]. In particular, an efficient modulation of thrombin activity could be obtained by the combined use of NU172 and HD22_27mer. Here we present the crystal structure of the ternary complex, in which thrombin is sandwiched between NU172 at exosite I and HD22_27mer at exosite II. Even though the crystalline packing reduces the conformational mobility of the two aptamers, the comparison of the present model with those of the relative binary complexes has revealed small but significant clues of an inter-exosites cross-talk. To evaluate the actual influence of the crystal packing on the long-range communication between the two exosites, a molecular dynamics study of the ternary complex is in progress. Details will be discussed at the Meeting. [1] E. Di Cera J Thromb Haemost. 2007, 5, 196. [2] S. Krishnaswamy J Thromb Haemost. 2005, 3, 54. [3] A.D. Keefe, S. Pai, A. Ellington Nat Rev Drug Discov. 2010, 9, 537. [4] I. Russo Krauss, A. Merlino, A. Randazzo, E. Novellino, L. Mazzarella, F. Sica Nucleic Acids Res. 2012, 40, 8119. [5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonova, C.A. Mattia, F. Sica Int J Biol Macromol. 2018, 107, 1697. [6] R. Troisi, V. Napolitano, V. Spiridonova, I. Russo Krauss, F. Sica Nucleic Acids Res. 2018, 46, 12177. [7] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica Acta Cryst. D 2013, 69, 2403. [8] N.S. Petrera, A.R. Stafford, B.A. Leslie, C.A. Kretz, J.C. Fredenburgh, J.I. Weitz J Biol Chem. 2009, 284, 25620. [9] X. Feng, C. Yu, F. Feng, P. Lu, Y. Chai, Q. Li, D. Zhang, X. Wang, L. Yao Chemistry 2019, 25, 2978. [10] K. Derszniak, K. Przyborowski, K. Matyjaszczyk, M. Moorlag, B. de Laat, M. Nowakowska, S. Chlopicki Front Pharmacol. 2019, 10, 68. [11] A. Pica, I. Russo Krauss, V. Parente, H. Tateishi-Karimata, S. Nagatoishi, K. Tsumoto, N. Sugimoto, F. Sica Nucleic Acids Res., 2017, 45, 461.