Abstract
Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.
Highlights
In eukaryotic organisms, phosphorylation is a common mechanism that regulates the activity of proteins involved in a large number of signaling pathways
Two conserved hydrophobic motifs, composed by non-consecutive residues and anchored to the αF-helix, are responsible for the correct positioning of the ATP molecule, the protein substrate, and the catalytic residues: the catalytic spine (C-spine), completed by the adenine ring of ATP, and the regulatory spine (Rspine), which is misaligned in protein kinases (PKs) inactive conformations.[3,4]
Even if PCA and classical Multidimensional Scaling (cMDS) methods can return the same results in specific contexts, cMDS can be considered as a more general method that maintains its validity in a rigorous sense for non-euclidean distances as Root Mean Squared Deviation (RMSD), i.e., the metric chosen in this study
Summary
Phosphorylation is a common mechanism that regulates the activity of proteins involved in a large number of signaling pathways. The transfer of the γ-phosphate from ATP to a given protein substrate is catalyzed by protein kinases (PKs) These proteins constitute about 2% of all human genes and their tight regulation is responsible for the correct development and maintenance of eukaryotic organisms.[1,2] As a result of their pivotal roles, PKs are exposed to several layers of control that encompass allosteric effectors, post-translational modification, and alteration of sub-cellular localization.[2,3]. Two conserved hydrophobic motifs, composed by non-consecutive residues and anchored to the αF-helix, are responsible for the correct positioning of the ATP molecule, the protein substrate, and the catalytic residues: the catalytic spine (C-spine), completed by the adenine ring of ATP, and the regulatory spine (Rspine), which is misaligned in PK inactive conformations.[3,4]
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