The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles - PubMed (original) (raw)

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Reiko Honda et al. EMBO J. 2005.

Abstract

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.

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Figures

Figure 1

Schematic diagram of cyclin E1 showing the relative positions of the nuclear localisation sequence (NLS) residues 12RSRKRK (Moore et al, 2002), the major site of phosphorylation (Thr380) and other phosphorylation sites (Thr62, Ser372 and Ser384 shown as bars) that are required for targeting cyclin E for ubiquitination and turnover (Welker et al, 2003), the construct used for the crystals of pCDK2/cyclin E1 (residues 81–363) and the positions of the N- and C-terminal cyclin box folds.

Figure 2

pCDK2/cyclin E1 and pCDK2/cyclin A complexes. pCDK2/cyclin E1 is shown in the top panel with pCDK2 in green and cyclin E1 in cyan. The activation segment of pCDK2 and the α1′/α2′ loop of cyclin E1 are labelled. pCDK2/cyclin A is shown in the lower panel with pCDK2 in yellow and cyclin A in magenta. Secondary structural elements are labelled. It is apparent that cyclin E1 makes more contacts to pCDK2 that involve the α1′/α2′ loop than does cyclin A.

Figure 3

Structures of cyclins E1 and A. The diagrams are colour coded following the spectrum (blue to red) from α1 to α5 of the N-terminal cyclin box fold and then from α1′ to α5′ of the C-terminal cyclin box fold. The N- and C-terminal helices are shown in white. The N-terminal cyclin box folds of cyclin E1 and cyclin A are very similar but there are significant differences in the C-terminal cyclin box folds. For further details, see text.

Figure 4a

Sequence alignments showing residues in contact at the pCDK2/cyclin interfaces. (A) Cyclin E1 and cyclin A aligned on the basis of structure for the construct used for cyclin E1 expression (residues 81–363). The secondary structure elements for cyclin E1 are indicated. Residues coloured in cyan from cyclin E1 are in contact with pCDK2 in the pCDK2/cyclin E1 complex and residues coloured in magenta from cyclin A are in contact with pCDK2 in the pCDK2/cyclin A complex. Contact is defined as any atoms <3.5 Å in separation from cyclin and pCDK2. Every 10th residue is marked with a line for cyclin E1.

Figure 4b

(B) Sequences of CDK2 showing secondary structural elements and CDK1. Residues coloured in green are in contact with cyclin E1 in the pCDK2/cyclin E1 complex and residues coloured in yellow are in contact with cyclin A in the pCDK2/cyclin A complex.

Figure 5

Details of the interactions between pCDK2/cyclin E1 and pCDK2/cyclin A. pCDK2/cyclin E1 is shown in the top panel and pCDK2/cyclin A in the lower panel. The colour scheme is the same as in Figure 1. (A) Interactions with the C-(PSTAIRE) helix. The hydrophobic face at the end of the cyclin E1 or cyclin A α5 helix interacts with hydrophobic residues Ile49 and Ile52 on pCDK2. The interface is further stabilised with hydrogen bonds from pCDK2 Arg50 to main-chain carbonyl of cyclin E1 Leu187, an interaction that is equivalent to that in pCDK2/cyclin A. With cyclin E1, there are also hydrogen bonds from pCDK2 Glu57 to cyclin E1 Tyr112 and Lys108 that are not made in pCDK2/cyclin A, while in pCDK2/cyclin A, there is a hydrogen bond from pCDK2 Lys56 to cyclin A Asp305, a contact that is not made in pCDK2/cyclin E because of sequence changes. For further details, see text. (B) Contacts at the 40s loop preceding the C-helix and the 70s loop at the β4/β5 turn. In the cyclin E1 complex, pCDK2 Glu42 hydrogen bonds to the main-chain nitrogen of cyclin E1 Leu195 at the start of α4, while in pCDK2/cyclin A, this residue makes no contacts because of a difference in conformation of the 40s loop. The pCDK2 main-chain carbonyls of Glu42 and Val44 contact cyclin E1 Lys186. Lys186 is localised by an ion pair with Glu215. At the pCDK2/cyclin A interface, Val44 main-chain carbonyl oxygen contacts Lys288, which in turn forms an ion pair with Glu295. In the pCDK2/cyclin A complex, pCDK2 Glu40 contacts cyclin A Lys289 and pCDK2 Thr41 main-chain carbonyl receives a hydrogen bond from cyclin A Lys289. These contacts are not possible in cyclin E1 because of sequence changes. At the β4/β5 turn (the 70s loop) in the pCDK2/cyclin A complex, there are contacts from pCDK2 His71 side chain and Thr72 main-chain oxygen to cyclin A His296 from the α5 helix. Because of sequence changes, the 70s loop adopts a different conformation and His71 interacts with Lys220. (C) Contacts at the pCDK2 activation segment and the α1′/α2′ loop of cyclin E1. The contacts to the phospho-Thr160 are described in the text. The α1′/α2′ loop makes additional interactions. The main chain oxygen of pCDK2 Val 154 hydrogen bonds to cyclin E1 Val237 (α1′ helix) main-chain nitrogen. Leu251 main-chain oxygen hydrogen bonds to the main-chain nitrogen of pCDK2 Val156 and Leu251 side chain makes van der Waals interactions with the pCDK2 residues Phe152 and Val156. These hydrophobic interactions are supported by cyclin E1 Trp95 from the region before the N-terminal helix, a region with a different conformation in cyclin E1 from that in cyclin A. For further details, see text.

Figure 6

Structure of pCDK2 (in green) in complex with cyclin E1 (in cyan) with the 20-amino-acid sequence (residues 230–249) that are involved in centrosome localisation (Matsumoto and Maller, 2004) shown in red. The PSTAIRE (C-) helix and the activation segment, the two major regions of contact between pCDK2 and cyclin E1, are shown in magenta.

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The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles - PubMed (original) (raw)