View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Research Article 1279 Structure of human cyclin-dependent kinase inhibitor p19INK4d: comparison to known ankyrin-repeat-containing structures and implications for the dysfunction of tumor suppressor p16INK4a Roland Baumgartner1, Carlos Fernandez-Catalan1, Astar Winoto2, Robert Huber1, Richard A Engh1* and Tad A Holak1* Background: The four members of the INK4 gene family (p16INK4a, p15INK4b, Addresses: 1Max Planck Institute for Biochemistry, p18INK4c and p19INK4d) inhibit the closely related cyclin-dependent kinases D-82152 Martinsried, Federal Republic of Germany 2 CDK4 and CDK6 as part of the regulation of the G →S transition in the cell- and Department of Molecular and Cell Biology, 1 University of California Berkeley, CA 94720-3200, INK4a division cycle. Loss of INK4 gene product function, particularly that of p16 , USA. is found in 10–60% of human tumors, suggesting that broadly applicable anticancer therapies might be based on restoration of p16INK4a CDK inhibitory *Corresponding authors. function. Although much less frequent, defects of p19INK4d have also been E-mail: [email protected] [email protected] associated with human cancer (osteosarcomas). The protein structures of some INK4 family members, determined by nuclear magnetic resonance (NMR) Key words: cell cycle, CDK4 inhibitor, p19INK4d, spectroscopy and X-ray techniques, have begun to clarify the functional role of p16INK4a, structure p16INK4a and the dysfunction introduced by the mutations associated with Received: 9 April 1998 human tumors. Revisions requested: 5 June 1998 Revisions received: 30 July 1998 Results: The crystal structure of human p19INK4d has been determined at 1.8 Å Accepted: 31 July 1998 resolution using multiple isomorphous replacement methods. The fold of Structure 15 October 1998, 6:1279–1290 p19INK4d produces an oblong molecule comprising five approximately http://biomednet.com/elecref/0969212600601279 32-residue ankyrin-like repeats. The architecture of the protein demonstrates the high structural similarity within the INK4 family. Comparisons to other © Current Biology Ltd ISSN 0969-2126 ankyrin-repeat-containing proteins (GABPβ, 53BP2 and myotrophin) show similar structures with comparable hydrogen-bonding patterns and hydrophobic interactions. Such comparisons highlight the splayed β-loop geometry that is specific to INK4 inhibitors. This geometry is the result of a modified ankyrin structure in the second repeat. Conclusions: Among the INK4 inhibitors, the highest amino acid sequence conservation is found in the helical stacks; this conservation creates a conserved β-loop geometry specific to INK4 inhibitors. Therefore, in addition to models which predict that the conserved helix α6 is responsible for CDK inhibition, a binding mode whereby the loops of INK4 proteins bind to the CDKs should also be considered. A similar loop-based interaction is seen in the complex formed between the ankyrin-repeat-containing protein GABPβ and GABPα. This mode of binding would be consistent with the observation that p16INK4a is sensitive to deleterious mutations found throughout this tumor suppressor protein; these mutations probably destabilize the three-dimensional structure. Introduction inhibited by specific CDK inhibitors [1,2]. One of these Prior to cell division, a cell must reproduce a new copy of inhibitors, p19INK4d, belongs to the INK4 family of CDK each of its chromosomes. This process occurs during a inhibitors, which are specific for kinases CDK4 and specific part of interphase, termed the DNA synthesis (or CDK6. CDK inhibition by INK4 inhibitors represents one → S) phase. The part of the cell cycle preceding S phase is of several stop mechanisms in the G1 S transition control referred to as G1. In the eukaryotic cell cycle, passage point, which ultimately controls progression to DNA through the restriction point and beginning of DNA syn- duplication. By inhibiting CDK4 and CDK6, the INK4 → thesis (i.e. the G1 S transition) is controlled by cyclins proteins can block CDK activity, prevent retinoblastoma and cyclin-dependent kinases (CDKs). Cyclin–CDK com- protein phosphorylation and the concomitant activation of plexes are activated by the phosphorylation of critical E2F transcription factors [1,2], and thereby induce G1 threonine and tyrosine residues of the CDKs and are phase cell-cycle arrest. The INK4 family comprises four 1280 Structure 1998, Vol 6 No 10 proteins: p16INK4a, p15INK4b, p18INK4c and p19INK4d [1–3]. structures and other biochemical information. Finally, in The most prominent member of the INK4 protein family order to highlight the distinguishing features of the INK4 is the multiple tumor suppressor protein p16INK4a [3–5]. It fold, we compare the structure of p19INK4d with other is believed that p16INK4a dysfunction and the disruption of INK4 protein structures and the other recently solved → the G1 S transition checkpoint may be required for the structures of ankyrin-repeat-containing proteins. genesis of many, or even most, human cancers [1,3]. In contrast to p16INK4a, other variant INK4 inhibitors are less Results and discussion frequently associated with cancer: no p18INK4c variant has Tertiary structure been associated with tumorigenesis [6]; one defective The crystals of p19INK4d contained a single molecule in p19INK4d variant is associated with cell lung cancer [7]; and the asymmetric unit. Interpretable electron density was several p15INK4b variants have been found in different obtained for residues 7–162 of p19INK4d; residues 1–6, human cancers [8]. Each member of the INK4 protein Glu129 and 163–166 were apparently disordered. family contains several 32-amino-acid ankyrin-like motifs p19INK4d forms an approximate ellipsoid with dimensions that form helix-turn-helix structures [9]. The INK4 55 Å × 25 Å × 20 Å. Figure 1 shows the striking topology inhibitors are highly homologous, for example, human of the highly α-helical p19INK4d with its five almost p19INK4d shares 48% sequence identity with that of human equally spaced helix-turn-helix segments between p16INK4a over a stretch of 130 amino acids [10]. residues 9–29, 54–62, 77–95, 110–128 and 142–159. With the exception of the second segment (residues 54–62), Here, we report the crystal structure of human p19INK4d each segment is 16–19 residues long and has a short turn [10,11] at 1.8 Å resolution. Together with a recently pub- located between the two helices. All of the β turns in the lished high-resolution X-ray structure of human p18INK4c helix-turn-helix segments have a central glycine residue [12] and NMR-derived structures of mouse p19INK4d [13] flanked by two residues in β conformations. The crystal and p16INK4a [14], the structure of human p19INK4d con- structure confirms a prediction, derived from NMR tributes to our understanding of the correlation between studies, of the secondary structure of ankyrin repeats, structure and mutational changes involved in tumorigene- which indicated that a typical ankyrin repeat should have sis in the INK4 inhibitors. Residues thought to be an eight to nine residue long hydrophobic α helix (start- involved in the interactions of the INK4 proteins with ing at residue 16) preceded by a glycine-containing CDK4 and CDK6 are also discussed on the basis of the β turn [15]. Figure 1 Stereoview Cα trace of p19INK4d (red) in a standard orientation superposed with p18INK4c (blue). Secondary structure elements and the N-terminal residue of each inhibitor (Arg7 and Trp5) are labelled. Research Article Structure and binding of p19INK4d Baumgartner et al. 1281 Figure 2 The primary sequence of human p19INK4d 1 10 20 30 (p19h) and a structural alignment with related Ankyrin X G X T P L H L A A R X G H V E V V K L L L D X G A D V N A X T K proteins. The aligned sequences are from consensus A I S Q N N L D I A E V K N P D D p18INK4c, p16INK4a (p15INK4b is not shown sequence V K T M R Q S I N E because of its very high identity, 75%, to p16INK4a), GABPβ, myotrophin and 53BP2. α1 α2 Residues that are identical in either all four 10 20 30 p19h 7 R A G D R L S G A A A R G D V Q E V R R L L H R E L V H P D A L N R INK4 family members or all seven proteins p18h 5 W . G N E L A S A A A R G D L E Q L T S L L Q N N . V N V N A Q N G within one ankyrin repeat are marked in bright p16h 12 S A D . W L A T A A A R G R V E E V R A L L E A G . A L P N A P N S yellow; conservation of residue type is marked GABPβ 5 D L G K K L L E A A R A G Q D D E V R I L M A N G . A P F T T . D W in light yellow. The boxed vertical yellow bars Myotrophin 1 M C D K E F M W A L K N G D L D E V K D Y V A K G . E D V N R T L E highlight residues that are absolutely 53BP2 324 N P L A L L L D S S L E G E F D L V Q R I I Y E . V D D P S L P N D conserved in all ankyrin repeats. Red cylinders α3 α4 indicate the α helices of p19INK4d (numbering 50 60 70 is as described in the text); the red arrows p19h 41 F G K T A L Q V M .