Human α-thrombin is a trypsin-like serine protease endowed with the unique ability to convert soluble fibrinogen in insoluble fibrin clot. In addition to the active site, this enzyme owns two electropositive regions, exosite I and II, located at opposite sides of its surface [1]. The narrow substrate specificity of thrombin and the ability to change function are regulated by the binding of different cofactors and modulators to the two exosites [2]. A special class of thrombin exosite synthetic ligands is represented by anticoagulant aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity [3]. The minimal 15mer DNA aptamer, named TBA, adopts a G-quadruplex structure and binds the fibrinogen-binding site of thrombin (exosite I) by a pincer-like recognition mechanism [4]. The addition of a duplex motif to the G-quadruplex module has produced a new generation of aptamers [5] with higher affinity against thrombin compared to TBA. Among them, HD22_27mer recognizes exosite II [6] and NU172, that is currently in clinical trials, binds thrombin exosite I [7]. In the last years, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on the two exosites of thrombin. In particular, biochemical studies have suggested an allosteric linkage between the two exosites [8]. Furthermore, insightful atomic-level snapshots of the concurrent binding of TBA and HD22_27mer to thrombin have been achieved through crystallographic analyses [9]. Recently, we have performed an extensive molecular dynamics analysis [10] on free thrombin and on its binary and ternary complexes with TBA and/or HD22_27mer in the absence of the PPACK inhibitor that is covalently bound to the protein active site in all the crystallographic α-thrombin models. Moreover, the structural and dynamic aspects of the simultaneous binding of NU172 and HD22_27mer to thrombin have been characterized. These studies have revealed that, in the absence of any influence of the crystal packing, an inter-exosite cross-talk occurs in these systems.

Simultaneous binding of aptamers at the two exosites of thrombin: structural and dynamics studies

Sica F.
2020

Abstract

Human α-thrombin is a trypsin-like serine protease endowed with the unique ability to convert soluble fibrinogen in insoluble fibrin clot. In addition to the active site, this enzyme owns two electropositive regions, exosite I and II, located at opposite sides of its surface [1]. The narrow substrate specificity of thrombin and the ability to change function are regulated by the binding of different cofactors and modulators to the two exosites [2]. A special class of thrombin exosite synthetic ligands is represented by anticoagulant aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity [3]. The minimal 15mer DNA aptamer, named TBA, adopts a G-quadruplex structure and binds the fibrinogen-binding site of thrombin (exosite I) by a pincer-like recognition mechanism [4]. The addition of a duplex motif to the G-quadruplex module has produced a new generation of aptamers [5] with higher affinity against thrombin compared to TBA. Among them, HD22_27mer recognizes exosite II [6] and NU172, that is currently in clinical trials, binds thrombin exosite I [7]. In the last years, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on the two exosites of thrombin. In particular, biochemical studies have suggested an allosteric linkage between the two exosites [8]. Furthermore, insightful atomic-level snapshots of the concurrent binding of TBA and HD22_27mer to thrombin have been achieved through crystallographic analyses [9]. Recently, we have performed an extensive molecular dynamics analysis [10] on free thrombin and on its binary and ternary complexes with TBA and/or HD22_27mer in the absence of the PPACK inhibitor that is covalently bound to the protein active site in all the crystallographic α-thrombin models. Moreover, the structural and dynamic aspects of the simultaneous binding of NU172 and HD22_27mer to thrombin have been characterized. These studies have revealed that, in the absence of any influence of the crystal packing, an inter-exosite cross-talk occurs in these systems.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11588/834831
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