Mapping the conformational energy landscape of Abl kinase using ClyA nanopore tweezers

Abstract

Protein kinases play central roles in cellular regulation by catalyzing the phosphorylation of target proteins. Kinases have inherent structural flexibility allowing them to switch between active and inactive states. Quantitative characterization of kinase conformational dynamics is challenging. Here, we use nanopore tweezers to assess the conformational dynamics of Abl kinase domain, which is shown to interconvert between two major conformational states where one conformation comprises three sub-states. Analysis of kinase-substrate and kinase-inhibitor interactions uncovers the functional roles of relevant states and enables the elucidation of the mechanism underlying the catalytic deficiency of an inactive Abl mutant G321V. Furthermore, we obtain the energy landscape of Abl kinase by quantifying the population and transition rates of the conformational states. These results extend the view on the dynamic nature of Abl kinase and suggest nanopore tweezers can be used as an efficient tool for other members of the human kinome.

Application of Nanopore Sensors for Biomolecular Interactions and Drug Discovery

“…[23] In 2022, Li et al used ClyA-AS mutant nanopores to investigate the morphological changes in the Abelson (Abl) kinase domain in the presence of the ATP-binding site inhibitor imatinib. [21] They confirmed two current states (S1: lobe-closed and S2: lobe-open) derived from the Abl kinase trapped within the ClyA pores (Figure 1C). They gave positive charges at the Nterminus of Abl in order to increase the residential time of Abl within the pore for monitoring the conformational dynamics.…”

“…In 2022, Li et al. used ClyA‐AS mutant nanopores to investigate the morphological changes in the Abelson (Abl) kinase domain in the presence of the ATP‐binding site inhibitor imatinib [21] . They confirmed two current states (S1: lobe‐closed and S2: lobe‐open) derived from the Abl kinase trapped within the ClyA pores (Figure 1C).…”

“…K D is the dissociation constant of imatinib against Abl kinase. Reproduced with permission [21] . Copyright 2022 The Author(s).…”

“…[18] Nanopore sensors can be applied for real-time analysis of enzyme at the single-molecule level. Several techniques of single-molecule enzyme analysis have been demonstrated using various biological nanopores, [19][20][21] wherein ligand binding and enzyme conformational dynamics have been observed at the single-molecule level. Although monitoring enzymatic reactions with nanopores is not fully established, nanopore enzymology, [22] the study of enzymes using nanopores, is attracting much attention.…”

Single-molecule fingerprinting of protein-drug interaction using a funneled biological nanopore

“…Previously, it was shown that small-molecule ligand bindings of trapped proteins could be detected using cylindrical ClyA nanopores 17 , 18 , 38 . In a ClyA nanopore, the ligand-induced conformational change of protein results in movement in its residence site within the pore, thereby inducing differential residual currents 16 .…”