Why Are the Truncated Cyclin Es More Effective CDK2 Activators than the Full-Length Isoforms? Soumya Lipsa Rath and Sanjib Senapati* Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India * S Supporting Information ABSTRACT: Cell cycle regulating enzymes, CDKs, become activated upon association with their regulatory proteins, cyclins. The G1 cyclin, cyclin E, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In vivo and in vitro studies have shown that these LMW isoforms of cyclin E hyperactivate CDK2 and accelerate the G1-S phase of cell division. The molecular basis of CDK2 hyperactivation due to LMW cyclin E isoforms in cancer cells is, however, unknown. Here, we employ a computational approach, combining homology modeling, bioinformatics analyses, molec- ular dynamics (MD) simulations, and principal component analyses to unravel the key structural features of CDK2-bound full-length and LMW isoforms of cyclin E1 and correlate those features to their differential activity. Results suggest that the missing N- and C-terminal regions of the cyclin E LMW isoforms constitute the Nuclear Localization Sequence (NLS) and PEST domains and are intrinsically disordered. These regions, when present in the full-length cyclin E/CDK2 complex, weaken the cyclin-CDK interface packing due to the loss of a large number of key interface interactions. Such weakening is manifested in the decreased contact area and increased solvent accessibility at the interface and also by the absence of concerted motions between the two partner proteins in the full-length complex. More effective packing and interactions between CDK2 and LMW cyclin E isoforms, however, produce more efficient protein-protein complexes that accelerate the cell division processes in cancer cells, where these cyclin E isoforms are overexpressed. C yclin dependent kinase 2 (CDK2) is a member of the eukaryotic serine/threonine protein kinase family that assists the phosphoryl transfer of ATP γ-phosphate to peptide substrates belonging to downstream targets. 1,2 CDK2 partic- ipates in cell cycle regulation at the G1/S boundary. 1,3 Its deregulation has been reported to associate with several human tumors, evoking strong interest to understand the molecular mechanism of CDK2 activation. 3,4 The recent structural and mutational studies have suggested that the full activation of CDK2 requires both binding of cyclin A/cyclin E and phosphorylation of residue T160 in the activation segment. 5-7 In the absence of these features, it has negligible activity because the residues that form the catalytic and substrate-recognition sites are disordered. 5,6 CDK2 contains 298 residues with a small N-terminal domain (residues 1-82), a large C-terminal domain (residues 88-298), and a short hinge region (residues 83-87) connecting the N- and C-terminal domains. 8 The N-terminal domain is primarily composed of a β-sheet, containing five antiparallel β-strands, and one α-helix. This helix with the “PSTAIRE” sequence (residues 45-56) is a signature of this class of proteins and constitutes the main point of interaction with cyclins. 8,9 The loop which precedes the PSTAIRE helix, known as the 40s loop (residues 33-41), also interacts with cyclins. The C-terminal domain is predominantly α-helical and contains the activation segment, the so-called T-loop (residues 145-172). The specific residue Thr160 in the T-loop undergoes phosphorylation by cyclin activating kinase (CAK) for complete CDK2 activa- tion. 7-9 The catalytic cleft that binds ATP is located between the N- and C-terminal domains. 6-9 A glycine rich loop, commonly known as G-loop, lies above the ATP binding pocket and is conserved in many kinases. 8 The primary function of this loop is to align the substrate and ATP correctly for a smooth transfer of the phosphate. 8,9 As a member of the CMGC group of kinases, CDK2 has a CMGC-specific structural feature, such as the CMGC insert at the CDK2- cyclin interface. 2 Cyclin E is the regulatory subunit of CDK2 in the late G1 phase. 10 The isoform, cyclin E1, contains 395 amino acid residues and is expressed in most proliferating normal and tumor cells. 10,11 The structure of cyclin E1 is primarily α-helical Received: April 4, 2014 Revised: June 20, 2014 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A dx.doi.org/10.1021/bi5004052 | Biochemistry XXXX, XXX, XXX-XXX