Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2

Pasquale Pisani, Fabiana Caporuscio, Luca Carlino, Giulio Rastelli, Claudio M Soares
2016 PLoS ONE  
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
more » ... e 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-1napthalenesulfonic 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. In eukaryotic organisms, 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] The fold of PKs is conserved throughout the whole family. [1] The naming convention based on the structure of the well-characterized cAMP dependent protein kinase (PKA) will be followed hereafter. [1, 4] The fold is organized around a large hydrophobic helix (αF) and consists of a small N-terminal (N-lobe) and a larger C-terminal lobe (C-lobe). The N-lobe is formed by five antiparallel β-strands (β1-β5) coupled to the so-called αC-helix and contains two conserved embedded sequences: the Glycine-rich Loop and the AxK motif. The C-lobe has a high helical content (αD-αI) and contains four helices that compose the hydrophobic core (αD, αE, αF, and αH), [4, 5] the PK catalytic machinery, including the so-called Catalytic Loop, and the highly conserved HRD and DFG motifs. The Asp of the DFG motif is responsible for the recognition of one of the ATP-bound Mg 2+ ions. The Activation Loop (A-loop), which is positioned between the DFG and a third conserved motif, APE, is one of the most variable regions of PKs and is involved in substrate binding. The two lobes of PKs are connected by a unique short loop known as the "hinge region". The phosphoryl transfer occurs in the deep cleft between the N and C-lobe. The relative positioning of the lobes influences the switch among the different conformational states. In particular, 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] With regard to their function, PKs can be depicted as molecular switches that can exist in an "on" state, which is maximally active, and different inactive states. All PKs that have been crystallized in the active form share common features. The Lys residue of the AxK motif bridges to a conserved Glu in the αC-helix. This salt bridge stabilizes the α and β phosphate groups of ATP. [2] The A-loop adopts an extended conformation that allows the binding of the substrate and the catalysis, spine residues are correctly aligned and the key catalytic residues are in the correct position to bind ATP.[6-9] On the contrary, the crystal structures of inactive PKs show a greater conformational heterogeneity. [3, 10] For example, the catalytic Asp of the DFG motif can adopt, at least, two distinct conformations: i) a DFG-in conformation, competent for the recognition and binding of ATP-bound Mg 2+ ions; and ii) an inactive DFG-out conformation, in which the positions of Asp and Phe are flipped, resulting in an extended ATP binding pocket. Both DFG-out and DFG-in inactive conformations (also known as Src-like inactive conformations) have been largely investigated. [10] [11] [12] In the former the Lys-Glu salt bridge is maintained, whereas in the latter the αC-helix is displaced compared with the active state, the Lys-Glu salt bridge is lost, and the R-spine is disrupted. Moreover, in the Src-like inactive form, the A-loop is collapsed over the active site, thus blocking the access of both the nucleotide and the substrate. PKs are targets of significant pharmaceutical interest. Inhibitors developed in the last decades can be classified according to their binding site. [13, 14] Type I and I½ inhibitors compete with ATP for the nucleotide binding site. However, the high sequence and structural conservation of the ATP binding site hampers the achievement of drug selectivity, thus rising New Metastable States of CDK2
doi:10.1371/journal.pone.0154066 pmid:27100206 pmcid:PMC4839568 fatcat:r5246wbdanarbe6q4pnsarxaya