Structure of Human Cyclin-Dependent Kinase Inhibitor P19ink4d

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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 . M F G S T A I A L E L L K Q G A . S P N V Q D . T indicate β turns. Red underlines indicate α- p18h 37 F G R T A L Q V M . K L G N P E I A R R L L L R G A . N P D L K D . R helical regions in the other proteins. An p16h 44 Y G R R P I Q V M . M M G S A R V A E L L L L H G A . E P N C A D P A ankyrin consensus sequence is given at the GABPβ 37 L G T S P L H L A A Q Y G H F S T T E V L L R A G S R D A R . . T . K top of the figure in blue [40]. Myotrophin 34 G G R K P L H Y A A D C G Q L E I L E F L L L K G A . D I N A P D . K 53BP2 359 E G I T A L H N A V C A G H T E I V K F L V Q F G V . N V N A A D . S

α5 α6 80 90 100 p19h 73 S G T S P V H D A A R T G F L D T L K V L V E H G . A D V N V P D . G p18h 69 T G F A V I H D A A R A G F L D T L Q T L L E F Q . A D V N I E D . N p16h 77 T L T R P V H D A A R E G F L D T L V V L H R A G . A R L D V R D . A GABPβ 70 V D R T P L H M A A S E G H A N I V E V L L K H G . A D V N A K D . M Myotrophin 67 H H I T P L L S A V Y E G H V S C V K L L L S K G . A D K T V K G . P 53BP2 392 D G W T P L H C A A S C N N V Q V C K F L V E S G . A A V F A M T Y S

α7 α8 110 120 130 p19h 106 T G A L P I H L A V Q E G H T A V V S F L A A E . . S D L H R R D A p18h 102 E G N L P L H L A A K E G H L R V V E F L V K H T A S N V G H R N H p16h 110 W G R L P V D L A E E L G H R D V A R Y L R A A A G G T R G S N H A GABPβ 103 L K M T A L H W A T E H N H Q E V V E L L I K Y . G A D V H T Q S K Myotrophin 100 D G L T A L E A T D N Q A I K A L L Q 53BP2 426 D M Q T A A D K C E E M E E G Y T T Q C S Q F L Y G V Q E K

α9 α10 140 150 160 p19h 138 R G L T P L E L A L Q R G A Q D L V D I L Q G H M V A P L p18h 136 K G D T A C D L A R L Y G R N E V V S L M Q A N G A G G A T N L Q p16h 144 R I D A A E G P S D I P D GABPβ 138 F C K T A F D I S I D N G N E D L A E I L Q Structure

The antiparallel helix-turn-helix motifs are connected to (Figure 2). The internal stability of a single helix-turn- neighboring repeats by long loops perpendicular to the helix segment is gained by the presence of several local long axis of the molecule. These loops (or fingers) are interaction motifs. Ankyrin repeats 2 and 3 clearly reveal a exposed to solvent and point away from the main body of C-terminal consensus sequence for helix capping in the molecule. They fold back onto the helical region by helices α4 and α6, which corresponds to the so-called forming type I β turns at segments 39–42, 71–74, 104–107 Schellman motif hxpxGh (where h = hydrophobic, and 136–139. We designate these loops as ‘β-hairpin-like’; p = polar, G = glycine and x = an arbitrary residue) they are called β sheets by others [12,13]. The helices [16–18]. Figure 2 shows the high degree of residue conser- from neighboring ankyrin repeats associate via hydropho- vation at these positions. Hydrogen bonds between back- bic interactions, such that each ankyrin pair forms a four- bone amide protons and carbonyl oxygen atoms were seen helix bundle. to link several residues: Ala65(N) to Leu60(O) and Gly64(N) to Leu61(O) in helix α4; Ala98(N) to Leu93(O) Local interactions stabilizing the secondary structure and Gly97(N) to Leu94(O) in helix α6. These hydrogen- The highly conserved residues among ankyrin repeats and bonding patterns are characteristic for the C-terminal helix among INK4 family members are responsible for either capping motif [17,18]. In addition, helices α5, α7 and α9 the stability of the ankyrin fold itself or for the stable possess an N-terminal proline residue that is known to packing between the ankyrin repeats — or for both facilitate helix formation [18]. 1282 Structure 1998, Vol 6 No 10

Figure 3

The conserved structural elements in ankyrin- repeat-containing proteins. (a) Stereoview illustrating the hydrogen bonds in loops 1 to 4 of p19INK4d, as an example of the hydrogen- bond network in the β loops of ankyrin-repeat- containing proteins. Hydrogen bonds shown in purple are formed by structurally equivalent residues of p18INK4c, GABPβ and 53BP2; hydrogen bonds in green are specific to only one of these proteins. The positions of hydrogen bonds were calculated using the program TURBO-FRODO [41]. Carbon atoms are shown in gray and nitrogen and oxygen atoms are shown in blue and red, respectively; sidechains not involved in hydrogen-bond networks are omitted from the figure. (b) Stereoview ribbon representation of p19INK4d showing details of the helix bundle formed by ankyrin repeats 3 and 4. The figure shows the hydrophobic core formed by residues Val94, Val100, Val122, Leu87, Leu90, Leu126 and Phe125. Sidechains of hydrophobic residues are shown in gray, nitrogen and oxygen atoms of basic and acidic amino acids are given in blue and red, respectively. Helices are shown in red and yellow.

The ankyrin repeat loop structures are stabilized by repeats 1, 2, 3 and 4, respectively (Figure 2), and bridge hydrogen bonds characteristic of β turns, and by intraloop peptide amide NH groups in the loop. In general, the hydrogen bonds formed by residues Asn39, Asp71, Asp104 stable stacking arrangement of ankyrin repeats can be and Asp136 (Figure 3a). These residues are strictly con- attributed to hydrogen-bond interactions between loops served at equivalent positions among INK4 ankyrin and hydrophobic interactions within helix bundles. For Research Article Structure and binding of p19INK4d Baumgartner et al. 1283

the loop stacking (Figure 3a), single mainchain hydrogen contains the conspicuously conserved sequence Gly-Phe- bonds link β turns 2 and 3, and β turns 3 and 4. Further- Leu-Asp-Thr-Leu and the β turns share several individual more, His79 and His112 bridge the loops by forming pairs conserved residues. of hydrogen bonds from the histidine ring to the two flanking loop backbones. His79 is strictly conserved The overall folds of human p16INK4a, p18INK4c and throughout the INK4 proteins, but His112 is replaced by p19INK4d and mouse p19INK4d, are very similar; however, aspartate in p16INK4a and p15INK4b. a comprehensive detailed comparison must await the availability of atomic coordinates for the NMR struc- The helices from neighboring ankyrin repeats pack by tures of the INK4 proteins. Figure 1 shows a superposi- aligning their hydrophobic surfaces. Figure 3b shows an tion of p19INK4d and p18INK4c: the overall backbone root example of the structural arrangement of amino acids mean square deviation (rmsd) is 2.0 Å. The folds are involved in such hydrophobic interactions. In this identical, except at the turns of helices α2 and α8 in example, a hydrophobic core is formed by residues Val94, ankyrin repeats 1 and 4, respectively (at the back in Val100, Val122, Leu90, Phe125 and Leu126. Although Figure 1). These differences correspond to an insertion only some of these residues are strictly conserved among in p19INK4d of one residue at position 32, compared to the INK4 proteins (Val122, Leu90 and Leu126), the sub- p18INK4c, and a deletion of two residues after position stitutions that do occur conserve the amino acid type 129. The deletion results in a relatively sharp turn at the (Val94→Leu/His, Val100→Leu, Phe125→Tyr). Figure 3b C-terminal end of helix α8 in p19INK4d (Figure 1). The also shows the distribution of polar or charged amino acids structure of p18INK4c was seen to exhibit two unusual that are exposed to solvent. features — a shortened helix α4 and a splayed arrangement of β loops 1 and 2. Both of these features also The second ankyrin repeat has an unusual feature at helix occur in the crystal structure of p19INK4d. Otherwise, the α3 (formed by residues 45–48 in p19INK4d and residues largest deviations are apparent in the β turns and at 41–44 in p18INK4c); in this repeat a short helical loop (α3) helix α9 of the last ankyrin repeat. The unusual feature replaces the usual eight-residue helix in the ankyrin of the second ankyrin repeat at helix α3 is found in all repeat helix-turn-helix motif. This difference is one of INK4 structures published so far [12–14], and was also several features that are associated with altered ankyrin noted in the NMR secondary structure determinations repeat stacking interactions. Another feature is the lack of of p19INK4d and p16INK4a [15,19]. In the p18INK4c struc- hydrogen bonding between the loops from ankyrin ture, a small cavity is found in this region of the protein repeats 1 and 2. In addition, two aromatic residues, Phe51 (Figure 4a; [12]) which, coupled with the high degree of and Phe86, are found at the interface between repeats 2 sequence conservation, led to the proposal that this and 3 such that the bulkier rings distend the helix-bundle could be a general feature of all INK4 proteins [12]. packing compared with the interactions described above This does not hold for our structure, however, as in (Figure 3b). This might also contribute to the ‘splayed’ p19INK4d the cavity is filled with two phenylalanine arrangement of β loops 1 and 2. sidechains (Figure 4).

Comparison to p18INK4c and other INK4 proteins The structure of the β loops is of particular interest in The INK4 inhibitors p15INK4b, p16INK4a, p18INK4c and terms of INK4 inhibitor functional studies, as the struc- p19INK4d exhibit a high degree of homology (Figure 2), tures of other ankyrin-repeat-containing proteins have sharing between 40% and 50% sequence identity (p18 and shown interactions with other proteins to occur via the cor- p19, 43% identity; p16 and p19, 48% identity over 130 responding loops (see discussion below). The geometry of residues; p16 and p18, 45% identity) [8,10,11]. The the splayed β loops 1 and 2, attributed to the shortened sequences of these inhibitors differ most obviously at the helix α3, seems likely to be a general feature of the C terminus: p15INK4b lacks the fifth ankyrin repeat, while inhibitors due to the conservation of the sequence at helix p16INK4a lacks both the C-terminal helix and an ankyrin α3. Furthermore, although highly flexible loops could be consensus sequence in the last 22 residues. The first four adapted to their crystalline environment, p19INK4d and the ankyrin repeats generally reflect the ankyrin consensus two p18INK4c monomers in the asymmetric unit are all very sequence [9], but each repeat also has specific features similar despite their diverse crystalline environments. characteristic of INK4 inhibitors (Figure 2). For example, Loops 1 and 2 are further apart in p19INK4d than in the first α helix in repeat 1 contains a strictly conserved p18INK4c; this might arise in part from the interposition of Ala-Ala-Ala-Arg-Gly segment, in the second repeat this is an aromatic sidechain from a symmetry-related molecule. shortened, in the third repeat this segment contains a Each of the two p18INK4c monomers also form crystal con- strictly conserved His-Asp-Ala-Ala-Arg sequence and in tacts via loops 1 and 2. Despite qualitatively different con- repeat 4 it is initiated with a Leu-Pro-h-x-Leu-Ala tacts, however, the highly similar loop conformations seen sequence. The overall similarities in the other segments in the crystal structure provide evidence for the observed are somewhat lower, although helix α6 of the third repeat loop arrangement in solution [13,14]. 1284 Structure 1998, Vol 6 No 10

Figure 4

The conformations of Phe51 and Phe86 in p19INK4d and their relationship to the p18INK4c structure. (a) Surface representation of p18INK4c and p19INK4d. Negatively charged residues are shown in red, positively charged residues are in blue. Yellow arrows indicate the cavity present in p18INK4c, but absent in p19INK4d. (The figure was prepared using the program GRASP [42].) (b) Stereoview of the

final 1.8 Å 2Fo–Fc electron-density map contoured at 1σ. The spatial arrangement of the two aromatic residues, Phe51 and Phe86, at the interface between ankyrin repeats 2 and 3 is shown.

A high-resolution structure is still lacking for p16INK4a. p16INK4a is less well known. NMR data showed the Nevertheless, the less precise NMR structures of p16INK4a segment of residues 134–156 to be disordered [14]. (and mouse p19INK4d), together with the higher precision However, truncation experiments removing residues after X-ray structures of p18INK4c and p19INK4d enable many position 131 abolished CDK binding. This truncation both structural characteristics of p16INK4a to be predicted with shortens the final helix α8 by one turn and removes the confidence. p16INK4a is folded into a stack of two full and C-terminal segment. If the abolished binding is not a two half ankyrin repeats [14]. The second repeat shares result of a disruption of the overall structure but is due to the shortened first α helix (α3) of p19INK4d and p18INK4c. the missing C terminus, the region might be expected to This observation alone strongly indicates the same adopt structure at least when bound to CDK. unusual splayed geometry of the first and second loops. Furthermore, the insertion of a proline residue (position Comparison to known ankyrin-repeat-containing structures 72 in p19INK4d numbering) lengthens the second loop in other than INK4 proteins p16INK4a. In addition, the p16INK4a NMR structure seems The structures of three ankyrin-repeat-containing pro- to indicate that the β turn is absent in loop 1 (Figure 2a of teins, other than the INK4 proteins, have been elucidated [14]). Thus, although p16INK4a shares with p18INK4c and so far [20–22]. The structure of the 53BP2 ankyrin domain p19INK4d the INK4 characteristic separation of loops 1 and bound to the p53 core domain and the structure of a 2, this might be exaggerated in the p16INK4a structure with ternary complex of GA-binding protein (GABP, a het- possibly a different local fold in loop 1. The p16INK4a helix erodimeric eukaryotic transcriptional regulator comprising stacking is likely to be very similar to that found in GABPα and GABPβ) bound to DNA were determined by p19INK4d; helices α5 and α6 have the most conserved X-ray crystallography; the structure of myotrophin was amino acid sequences among all INK4 proteins. The determined by NMR [20]. A detailed comparison of structure, or disorder, of the C-terminal segment of 53BP2 and p18INK4c was carried out by Venkataramani et Research Article Structure and binding of p19INK4d Baumgartner et al. 1285

Figure 5

Structural comparison of INK4 proteins with GABPβ and myotrophin. (a) Stereoview Cα atom superposition of p19INK4d (red) with GABPβ (blue). (b) Stereoview Cα atom superposition of p19INK4d (red) with myotrophin (blue).

al. [12], who showed that the overall multirepeat domain the two structures is the position of loop 1. In addition, the from the 53BP2 protein superimposes well with the α3 fragment of the second ankyrin repeat unit in the respective regions of the p18INK4c structure. The N-termi- INK4 proteins consists of just a single turn-helix, whereas nal GABPβ subunit of the GABPα–GABPβ heterodimer in GABPβ the complete helix-turn-helix is preserved. In consists of three full and two half ankyrin repeats. the ankyrin repeat of the GABPβ molecule, the helix adja- Comparison of p19INK4d with the structure of GABPβ also cent to the loop has mostly short residues, whereas the showed high similarities for these protein folds (Figure 5a). residues from the helix on the face opposite to the loop are The pairwise backbone rmsd between p19INK4d and longer. This asymmetry gives rise to a distinct curvature in GABPβ is 5.9 Å. The most significant difference between the packing arrangement of adjacent ankyrin-repeat 1286 Structure 1998, Vol 6 No 10

helices. p19INK4d and p18INK4c have a similar asymmetric The formation of hydrophobic cores between the helices of distribution of long and short residues, although less pro- two neighboring ankyrin repeats can be found in all the pro- nounced than in GABPβ. In the structure of the teins discussed, reflecting the high content of hydrophobic GABPβ–GABPα complex, the tips of the loops of GABPβ residues. In the first helix of each ankyrin repeat there is a form a concave surface that curves around one side of conserved alanine residue that probably serves to accommo- GABPα. Compared to the p19INK4d structure, the GABPβ date the larger leucine, isoleucine and valine residues pref- curvature arises primarily from the movement of loop 1 erentially located in the second helix. This alanine is absent closer towards loop 2 (Figure 5a). It is possible, however, in the short helix α3 of INK4 proteins but present in that this configuration of GABPβ is influenced by binding GABPβ, myotrophin and 53BP2, which possess a full seven to GABPα. to eight residue long helix at this position. Helices are initiated in most cases by typical helix-promoting residues. The Figure 5b shows a superposition of the p19INK4d structure C-terminal helix-capping motif found in repeats 2 and 3 of with the structure of myotrophin. Myotrophin is a p19INK4d is also present in the corresponding regions of 12.5 kDa protein that appears to have a key role in the ini- GABPβ, myotrophin and 53BP2. tiation of cardiac hypertrophy. The central part of the protein contains two full ankyrin-like repeats. The N ter- Binding modes of INK4 proteins to CDK4 minus comprises a well defined ankyrin repeat which, as The central question regarding the function of INK4 in GABPβ and the INK4 proteins, lacks the first N-termi- inhibitors concerns the geometry of CDK4/6 binding. nal β-like structure segment. The C terminus of Several studies have aimed to address this question and a myotrophin also consists of α-helical elements arranged variety of experimental data are available. Possibly the roughly into an ankyrin-like fold. A superposition of any of most important observation is that the four INK4 proteins the two central ankyrin-like repeats of myotrophin with appear to be biochemically indistinguishable with respect any of the repeat units of the p19INK4d structure (except to CDK4 versus CDK6 binding [8,10]. Additional impor- repeat unit 2) shows an rmsd between backbone atoms of tant data are provided by the structures of other ankyrin- 1.4 Å with a total overall rmsd of 10 Å. repeat-containing proteins, the effects of mutations on binding, and the results of peptide-binding studies. To It is obvious from inspection of Figure 2, that all of the date there are two published structures of ankyrin-repeat- proteins used in the sequence alignment possess the containing proteins in complex with their target proteins: ankyrin consensus sequence. The presence of identical or the 53BP2 ankyrin domain–p53 complex and a complex of conserved residues at specific positions reflects the impor- GABPα and GABPβ bound to DNA. The interaction of tance of these residues in maintaining the characteristic the 53BP2 ankyrin domain with p53 is mediated by structural topology of the ankyrin motif. In GABPβ and residues in the β-loop segments of the ankyrin repeats 53BP2, the hydrogen-bond network between the loop [21]. Similarly, GABPβ interacts with GABPα exclusively regions shows the same features as in the INK4 family through the tips of the four β loops of ankyrin repeats, by proteins (Figure 3a); intraloop hydrogen bonds between inserting them into a depression in GABPα. As described the sidechains of either threonine, aspartate or asparagine in the previous section, the first N-terminal β loop of and the loop backbone are present in exactly the same GABPβ (loop 1 in p19INK4d) is hydrogen bonded to the arrangement. The tips of the loops comprise the consen- next loop in GABPβ. This differs from the arrangement in sus sequence (Thr/Asp/Asn)-x-x-Gly which maintains the p19INK4d and p18INK4c, where helix α3 of the second hydrogen-bond network while allowing a possibly func- ankyrin repeat unit consists of just a single turn. As a tional hypervariability at the ‘x-x’ positions. result, the interactions between α3 and α5, and contacts between neighboring loops, are less extensive in this In p19INK4d, loops 2, 3 and 4 are bridged by His79 and segment of the structure than in the corresponding junc- His112 forming hydrogen bonds to both sides of the tions of other repeat units. By analogy with the inter- neighbouring loops (Figure 3a). In 53BP2, there are two protein interactions found in the 53BP2–p53 and histidine residues (His365 and His398) at structurally GABPα–GABPβ complexes, it would be reasonable to similar positions. These histidines are also present in postulate that the INK4 proteins also bind to the CDKs GABPβ together with a third histidine residue (His34) via their β-loops. It is possible that a plastic deformation bridging loops 1 and 2 in an equivalent way. In addition, might accompany such CDK binding, which would bring in GABPβ the first loop is integrated into the hydrogen- the loops closer together in the binding site, in an analo- bond network and not separated as in INK4 proteins. gous manner to GABPα–GABPβ binding. An ‘inter- Interloop hydrogen bonds can be found between domain’ movement around α3 would facilitate such a Leu37(O) and Lys69(N) and from Trp36(O) to the motion. On the other hand, the unusual INK4 β-loop sidechain of Lys69. The current precision of the NMR geometry might be required for CDK4/6 recognition. Evi- structure of myotrophin does not allow for accurate dence against this model includes the screening of small hydrogen-bonding comparisons. p16INK4a-derived peptides for binding to CDK4 and Research Article Structure and binding of p19INK4d Baumgartner et al. 1287

Figure 6

Summary of mutations that affect binding of (a) p16INK4a to CDK4/6. (a) The amino acid 20 30 40 sequence of p16INK4a is divided into its four R R A A A G E G D ankyrin repeats (shown boxed in gray). Mutations that abolish the binding of p16INK4a p16h12 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 to CDK4 are depicted in red letters, and GAAAAA those that do not significantly affect binding D are in cyan. Green letters indicate mutations 50 60 70 that reduce binding to less than 50%. Circles mark the surface-exposed residues. The data H T R T K P R Y S presented are based on results from p16h44 Y G R R P I Q V M M M G S A R V A E L L L L H G A E P N C A D P A [14,19,23,25–32]. (b) Stereoview of a A A G A T p16INK4a model showing the spatial location of the mutations listed in (a). Amino acid mutations that abolish the binding of p16INK4a 80 90 100 to CDK4/6 are shown as red spheres. All H P R other mutations (no effect or reduced activity) L NT AAA E P P P W are indicated by gray spheres. p16h denotes G the human p16INK4a sequence followed by the p16h77 T L T R P V H D A A R E G F L D T L V V L H R A G A R L D V R D A residue number. ANA S Q Y AV AM C

110 120 130

AL AA QHD p16h110 W G R L P V D L A E E L G H R D V A R Y L R A A A G G T R G S N H A S AAAA G

150 p16h144 R I D A A E G P S D I P D AKPT (b)

Structure

CDK6, which indicated that the helical region α5–α6 of binding to CDK4 and CDK6, diverse inhibition of CDK4 the third ankyrin repeat (residues 84–103 of p16INK4a) and cyclin D1 kinase activity in vitro, and blocked cell- comprised the primary CDK4-binding site [23,24]. These cycle progression through G1 [23]. Very recently, the same screening experiments tested a series of overlapping 20- authors were able to reduce the minimal CDK binding residue peptides that spanned the complete p16INK4a amino sequence to ten residues [24]. The ten-amino-acid acid sequence. Several appeared to mimic the binding of minimal CDK4 interacting segment is located between INK4 proteins to CDK4: they showed differentiated residues 89–99 in p16INK4a (residues 85–95 in p19INK4d; 1288 Structure 1998, Vol 6 No 10

that is, it contains a complete ten-residue sequence of that mutations are neither clustered within a specific helix α6). region of the protein nor are there any hot spots. The loca- tions of the mutations, regardless of whether they are The binding of p16INK4a to CDKs was also studied using deleterious or not, show no obvious correlation between C-terminal deletions of p16INK4a [25]. Eight deletion solvent-exposed sidechains and those buried inside the series were produced with truncation at residues Asp153, protein. It is clear, however, that most of the mutations Glu120, Trp110, Arg80, Glu69, Glu61 and Arg58 of the interfere with proper helix packing or preservation of the wild-type p16INK4a sequence. The only variant that hydrogen-bond network, but they may not directly partici- retained the ability to bind to CDK4 and CDK6 was the pate in binding to the CDKs. These mutations lead to segment truncated at residue 153. All the other trunca- defective folding of the protein and/or increased aggrega- tions resulted in loss of binding, including the Trp110 and tion, which ultimately leads to insoluble protein. For Glu120 fragments, which each contain three complete example, Zhang and Peng [27] clearly showed that the ankyrin repeats in addition to the ten-amino-acid p16INK4a mutants Asp74→Αsn, His87→Pro, His98→Pro, sequence implicated in the binding to CDK4 and CDK6. Val126→Asp, Pro81→Leu, Gly101→Trp and Pro114→Leu Similar results were obtained by Yang et al. who reported all have disrupted backbone folding with five of the seven that deletion to Arg131, or removal of the fourth ankyrin proteins associating to form multimers in solution. An repeat, abolished CDK binding [26]. At first sight, the interesting mutation in this group is Asp74→Asn. This rel- p16INK4a CDK peptide screening and the p16INK4a trunca- atively conservative amino acid substitution has a pro- tion data seem to contradict each other. One simple expla- nounced effect on the structural integrity and stability of nation might be that truncations lead to aggregation of p16INK4a. This is supported by our model structure of p16INK4a and thus elimination of CDK binding [25,27]. p16INK4a: in the model, substitution of Asp74→Asn removes the salt bridge between Arg46 and Asp74. Muta- Although the synthetic peptide screening suggests that tions found in familial melanoma provide another illustra- the CDK-binding region is located at helix α6, at this tion as to how a small disruption of the secondary structure stage, a model that assumes that the binding mode of the can have major effects on the function of p16INK4a [25]. INK4 inhibitors is analogous to those of 53BP2 and For example, based on the p18INK4c structure, it has been GABPβ proteins cannot be rejected. The answer to this suggested [29] that the Arg87→Pro mutation destabilizes puzzle can only be provided with the structure of the the entire structure by removing the salt bridge between binary INK4–CDK complex. Asp84 and Arg87. The corresponding salt bridge is also present in the structure of p19INK4d (Asp80–Arg83). In Implications for p16INK4a mutations conclusion, it now appears that most tumor-derived muta- Extensive biochemical data are currently available on the tions in p16INK4a globally destabilize the secondary struc- effects of mutations in p16INK4a, especially those muta- ture and sidechain packing of the entire p16INK4a molecule, tions that are found in human cancers [1,3,12,24–32]. To rather than locally disrupting the CDK4/6-binding site. date, three mechanisms that lead to tumor-associated inactivation of p16INK4a have been characterized. These Biological implications include point mutation, homozygous deletion and hyper- Cell division is controlled by a series of positive and neg- methylation of the CpG island spanning the promoter ative regulators that act at sequential points throughout region of the p16INK4a gene [3,29]. More than 160 mis- the cell-division cycle. Disturbance of these control sense mutations have been identified, and it now appears points contributes to cancer by allowing uncontrolled that deletion and frameshift mutations in p16INK4a are cell proliferation. During the cell-division cycle, cells in even more frequent [3,29]. It is now uniformly accepted interphase pass from a stage termed G1 to the DNA INK4a that the loss of p16 function (i.e. inhibition of the synthesis (or S) phase. At G1, cells irrevocably commit cyclin D–CDK4 complex activity or inhibition of entry to DNA synthesis; this point is controlled by protein into the S phase) is directly connected to the loss of complexes consisting of cyclin-dependent kinases CDK4/6 binding by the p16INK4a mutants [25,27,29]. We (CDK4 and CDK6) and cyclins D. These complexes are concentrate here on point mutations that abolish this inhibited by the INK4 family of proteins — p16INK4a, binding in vitro, and try to explain them on the basis of p15INK4b, p18INK4c and p19INK4d. Genetic alterations structural considerations. Figure 6 gives an overview of affecting p16INK4a and cyclin D1 are so frequent in the distribution of such mutations in the p16INK4a struc- human cancers that inactivation of these proteins is ture. It includes mutations that are not only identified in believed to be necessary for tumor development. tumors, but those which also reduce notably CDK4- Broadly applicable anticancer therapies might be based binding activity in vitro. Considering the model of on restoration of p16INK4a CDK inhibitory function. β-loop–CDK interactions presented above, the mutation Although found much less frequently than in p16INK4a, Tyr44→His is conspicuously exposed to solvent and defects of p19INK4d have also been implicated in human distant from helix α6 (Figure 6b). In general, it is evident cancer (osteosarcomas). Research Article Structure and binding of p19INK4d Baumgartner et al. 1289

We report here the three-dimensional structure of obtained by breaking up stacks and then soaking them in a harvesting human p19INK4d determined by X-ray crystallography. solution containing 35% PEG 4000, 0.1 M Tris/HCl, 0.2 M MgCl2 INK4d (pH 8.5). The crystals were monoclinic, with typical cell dimensions of The fold of p19 produces an oblong molecule built a = 28.4 Å, b = 54.4 Å, c = 45.6 Å. The crystals were in space group up from five approximately 32-residue ankyrin-like P21 with one molecule per asymmetric unit. The solvent content was repeats. The structure confirms the high structural simi- 35% resulting in a Matthews coefficient of 1.95. Heavy-atom deriva- larity within the INK4 family members, in particular a tives were prepared by conventional soaking experiments (Table 1). splayed β-loop geometry and modified ankyrin structure X-ray data collection in one repeat. Comparisons to other ankyrin-repeat-con- The diffraction data were collected in oscillation mode on a 30 cm taining proteins, including GABPβ, 53BP2 and image-plate detector (MAR Research, Hamburg, Germany) attached to myotrophin, show similar structures with specific hydro- a Rigaku RU200 rotating-anode generator providing graphite mono- α gen-bonding patterns and hydrophobic interactions. chromatized Cu-K radiation at 7°C. The oscillation range was 2 β degrees resulting in near 100% completeness within 90 frames. The Such comparisons highlight the splayed -loop geometry statistics for native and heavy-atom derivative data are given in Table 1. that is specific to INK4 inhibitors. This unusual geome- Data were indexed, merged and reduced with the DENZO/- try results from the modified ankyrin structure in the SCALEPACK software package [33]. Very low values for the distortion second repeat. matrix were obtained for both monoclinic and orthorhombic lattice types resulting in possible space groups P2 and P222, respectively. During refinement and data reduction, the lattice type and space group The highest sequence conservation among the INK4 clearly proved to be monoclinic P21, with good Rmerge values and spot inhibitors is found in the helical stacks, and this gener- rejection below 1%. ates the conserved, INK4-specific loop geometry. There- Structure determination by multiple isomorphous replacement fore, although models have been predicted in which Derivative data sets were scaled against the native set using CAD and helix α6 is responsible for CDK inhibition, a binding SCALEIT of the CCP4 [34] program suite. Heavy-atom positions were mode whereby the loops of INK4 proteins bind to CDK4 determined with the program RSPS [35] and refined using a vector- and CDK6 should also be considered. A similar loop- space refinement of the heavy-atom difference Patterson functions based interaction is seen in the interaction between the implemented in VECREF [34]. Additionally, these positions could be visually identified in Harker sections of difference Patterson maps at β α ankyrin-repeat-containing protein GABP and GABP . different resolutions. All derivatives were analyzed by cross-difference This mode of binding would be consistent with the Fourier maps and referred to the same origin. In the next round, a observation that p16INK4a is sensitive to deleterious maximum-likelihood refinement, as implemented in the program mutations found throughout the tumor suppressor MLPHARE [34], was used for positional refinement and phase calcula- tion. The electron density was modified by applying solvent flattening protein, rather than to a cluster of mutations near a and histogram matching options in the DM program. The high quality of single binding region. These mutations are most likely to the resulting density clearly showed the molecular envelope and sym- lead to the destabilization of the three-dimensional struc- metry-related molecules. Model building was performed from an initial ture of the INK4 proteins. skeletonization of the electron density using MAIN [36]. Three helices were identified in the initial skeleton serving as a starting point. With the exception of six N-terminal residues, four C-terminal residues, and Materials and methods residue Glu129, the complete polypeptide chain could be traced. Protein purification and crystallization Rigid-body refinement was carried out using X-PLOR [37] confirming The stock solution of human p19INK4d at a concentration of 15 mg/ml the positional arrangement and unit-cell packing of the model. Several was prepared as described previously [13]. Crystals were grown at rounds of interactive model building and refinement with REFMAC [38] 4°C using sitting drops equilibrated against a reservoir of 30% polyeth- produced a crystallographic R factor and Rfree of 19.6% and 26.4%, ylene glycol (PEG) 4000, 0.1 M Tris/HCl, 0.2 M MgCl2 (pH 8.5). Drops respectively. The resolution of the final model was 1.8 Å. Following the (6 µl) were set up at a 1:1 ratio of reservoir to protein solution. Crystals determination of the p18INK4c structure [12], molecular replacement started to grow from the precipitate as stacks of thin plates after 1.5 using AMORE [39] with standard protocols proved successful using months and grew to their maximum size over a period of three weeks. the p18INK4c coordinates, but failed using the 53BP2 ankyrin-repeat Single crystals of suitable size, typically 0.1 × 0.1 × 0.2 mm, were structure [21]. The refinement statistics are summarized in Table 2.

Table 1

Data collection statistics.

† Data set No. of unique Resolution Phasing power Completeness RCullis Rmerge Soaking reflections (Å) (acentric)* (%) (total) (concentration/ time)

Native 13 017 1.8 — 99.4 — 0.073 —

UO2(CH3CO2)2 5462 2.4 2.96 98.6 0.51 0.085 2.5 mM/ 5 days Na2WO4 7019 2.2 2.92 99.5 0.52 0.108 5.0 mM/ 3 days EuCl3 6636 2.2 0.27 94.4 0.98 0.088 2.5 mM/ 23 days HgCl2 5058 2.4 3.01 85.4 0.79 0.092 5.0 mM/ 3 days NaAuCl4 6753 2.2 2.98 96.0 0.51 0.082 5.0 mM/ 3 days

Σ 2 Σ 2 1/2 † Σ Σ Ι Ι Σ Σ *Limiting resolution 3 Å, except for EuCl3 (5 Å). Phasing power = [ hklFh / hkl (FPH,obs – FPH,calc) ] . Rmerge = hkl i | hkl,i – < hkl> | / hkl iIhkl. 1290 Structure 1998, Vol 6 No 10

Table 2 13. Luh, F.Y., et al., & Laue, E.D. (1997). Structure of the cyclin- dependent kinase inhibitor p19Ink4d. Nature 389, 999-1003. Data refinement statistics. 14. Byeon, I.-J., et al., & Tsai, M.-D. (1998). Tumor suppressor p16INK4a: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4. Mol. Cell 1, 421-431. No. of protein atoms 1167 15. Kalus, W., et al., & Holak, T.A. (1997). NMR structural characterization No. of water molecules 198 of the CDK inhibitor p19INK4d. FEBS Lett. 401, 127-132. R factor (%)* 19.6 16. Parker, M.H. & Hefford, M.A. (1997). A consensus residue analysis of † α Rfree (%) 26.4 loop and helix-capping residues in four- -helical-bundle proteins. Root mean square deviation§ Protein Eng. 10, 487-496. bonds (Å) 0.0058 17. Aurora, R. & Rose, G.D. (1998). Helix capping. Protein Sci. 7, 21-38. angles (°) 0.96 18. Blanco, F.J., Ortiz, A.R. & Serrano, L. (1997). Role of a nonnative 2 interaction in the folding of the protein G B1 domain as inferred from B factor (Å ) 3.2 conformational analysis of the α-helix fragment. Fold. Des. 2, 123-133. Average figure of merit# 0.78 19. Tevelev, A., et al., & Tsai, M.D. (1996). Tumor suppressor p16INK4a: Σ Σ † structural characterization of wild-type and mutant proteins by NMR *R factor = hkl | Fobs – Fcalc | / hklFobs. Rfree was calculated using 5% and circular dichroism. Biochemistry 35, 9475-9487. § of the reflections randomly omitted from the refinement. After final 20. Yang, Y., Sambasivarao, N., Sen, S. & Qin, J. (1998). The structural minimization. #After density modification. basis of ankyrin-like repeat function as revealed by the solution structure of myotrophin. Structure 6, 619-626. 21 Svetlana, G. & Pavletich, N.P. (1996). Structure of the p53 tumor INK4a p16 model building suppressor bound to the ankyrin and SH3 domains of p53BP2. The p16INK4a backbone conformation was modeled according to that of Science 274, 1001-1005. p19INK4d using the program O. The model comprises residues 22. Batchelor, A.H., Piper, D.E., de la Brousse, F.C., McKnight, S.L. & Ser12–Thr137. A two-residue insertion at p16INK4a position 132 (posi- Wolberger, C. (1998). The structure of GABPα/β: an ETS domain- tion 128 in p19INK4d) was built according to that found in p18INK4c. ankyrin repeat heterodimer bound to DNA. Science 279, 1037-1041. There are two residue deletions compared to p19INK4d — after p16INK4a 23. Fahraeus, R., Paramio, J.M., Ball, K.L., Lain, S. & Lane, D.P. (1996). positions 14 and 35 — and also one insertion — at position 75. These Inhibition of pRb phosphorylation and cell-cycle progression by a 20- residue peptide derived from p16CDKN2/INK4A. Curr. Biol. 6, 84-91. positions were modeled in the most likely residue conformations as 24. Fahraeus, R., Lain, S., Ball, K.L. & Lane, D.P. (1998). Characterization there are no similar regions in the available coordinate sets. Sidechain of the cyclin-dependent kinase inhibitory domain of the INK4 family as conformations of identical residues were taken from the p19INK4d struc- a model for a synthetic tumor suppressor molecule. Oncogene 16, ture. The most preferred sidechain conformation was assigned to 587-596. mutated residues. Finally, the model was energy minimized using the 25. Parry, D. & Peters, G. (1996). Temperature-sensitive mutants of p16CDKN2 program X-PLOR [37]. associated with familial melanoma. Mol. Cell Biol. 16, 3844-3852. 26. Yang, R., Gombart, A.F., Serrano, M. & Koeffler, H.P. (1995). Mutational effects on the p16INK4a tumor suppressor protein. Cancer Accession numbers Res 55 INK4d . , 2503-2506. The atomic coordinates for the p19 structure have been deposited 27. Zhang, B. & Peng, Z. (1996). Defective folding of mutant p16INK4 proteins with Brookhaven Protein Data Bank (accession code 1BD8). encoded by tumor-derived alleles. J. Biol. Chem. 271, 28734-28737. 28. Reymond, A. & Brent, R. (1995). p16 proteins from melanoma-prone Acknowledgements families are deficient in binding to CDK6. Oncogene 11, 1173-1178. We thank Ronen Marmorstein for providing the coordinates of p18INK4c, 29. McDonald, N.Q. & Peters, G. (1998). Ankyrin for clues about the NP Pavletich for the coordinates of 53BP2 and Jun Qin for the coordinates function of p16INK4a. Nat. Struct. Biol. 5, 85-88. of myotrophin. We thank Dusan Turk and Hans Georg Beisel for critical sci- 30. Ranade, K., et al., & Dracopoli, N.C. (1995). Mutations associated with entific discussions. familial melanoma impair p16INK4 function. Nat. Genet. 10, 114-116. 31. Arap, W., Knudsen, E.S., Wang, J.Y.J., Cavenee, W.K. & Huang, H-J.S. (1997). 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