Mechanism of p27 Unfolding for CDK2 Reactivation.

Soumya Lipsa Rath,Sanjib Senapati

doi:10.1038/srep26450

Abstract

Cell-cycle regulatory protein, CDK2 is active when bound to its complementary partner protein, CyclinA or E. Recent discovery of the Kip/Cip family of proteins has indicated that the activity of CDK2 is also regulated by a member protein, p27. Although, the mechanism of CDK2 inhibition by p27 binding is known from crystal structure, little is known about the mechanism of CDK2 reactivation. Here, we execute classical and accelerated molecular dynamics simulations of unphosphorylated- and phosphorylated-p27 bound CDK2/CyclinA to unravel the CDK2 reactivation mechanism at molecular-to-atomic detail. Results suggest that the phosphorylation of p27 Y88 residue (pY88-p27) first disrupts the p27/CDK2 hybrid β-sheet and subsequently ejects the p27 310 helix from CDK2 catalytic cleft. The unbinding of p27 from CDK2/CyclinA complex, thus, follows a two-step unfolding mechanism, where the 310 helix ejection constitutes the rate-limiting step. Interestingly, the unfolding of p27 leaves CDK2/CyclinA in an active state, where the prerequisite CDK2-CyclinA interfacial contacts were regained and ATP achieved its native position for smooth transfer of phosphate. Our findings match very well with NMR chemical shift data that indicated the flip-out of p27 310 helix from CDK2 pocket and kinetic experiments that exhibited significant kinase activity of CDK2 when saturated with pY88-p27.

Highlights

Cyclin Dependent Kinase 2 (CDK2) involves two major steps: (i) its association with the regulatory subunit – CyclinA or CyclinE and (ii) phosphorylation of the residue Thr1603–5
Recent studies have shown that the binding of p27 to CDK2 inhibits CDK2 activity, while phosphorylation of certain p27 tyrosine residues resumes the activity of CDK2
Even though NMR chemical shift data indicated the ejection of p27 310 helix from CDK2 catalytic site, the detailed mechanism of p27 unbinding from the inactive p27/CDK2/CyclinA ternary complex and subsequent mechanism of CDK2 reactivation are unknown

Summary

Introduction

CDK2 involves two major steps: (i) its association with the regulatory subunit – CyclinA or CyclinE and (ii) phosphorylation of the residue Thr1603–5. Recent discovery of the Kip/Cip and INK4 family of proteins has indicated that the activity of CDKs is regulated by this family of proteins, which bind to and inhibit the catalytic activity of CDKs for cell-cycle control[5,6]. The crystal structure of pCDK2/CyclinA bound to the kinase inhibitory domain of p27 was solved at 2.3 Å resolution (Fig. 1, from here referred as p27/CDK2/CyclinA). The crystallized kinase inhibitory domain of p27 contained 69 amino acids and is comprised of a rigid coil (residues 26–35), an amphipathic α-helix (residues 38–59), an amphipathic β-hairpin (residues 61–71), a β-strand (residues 75–81), and a 310 helix (residues 85–90). The position of β2 strand and that of the G-loop and β1 strand in active CDK2 is shown (in grey, labelled in grey) from the crystal structure of CDK2/CyclinA complex (PDB ID: 1QMZ28) by superposing the two crystal structures. The crystal structure of p27/CDK2/ CyclinA does not contain the ATP molecule, nor it contains the glycine rich loop, the so-called G loop, that aligns the substrate and ATP correctly for a smooth transfer of the γ-phosphate[11,17,18,19]

Methods

Results

Conclusion