Crystal structure of casein kinase-1, a phosphate ... - Europe PMC

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Dec 8, 1994 - 101 Waukegan Road, Lake Bluff, IL 60044 and 2Biology Department,. Brookhaven National Laboratory, Upton, NY 11973, USA.
The EMBO Journal vol.14 no.5 pp.1015-1023, 1995

Crystal structure of casein kinase-1, phosphate-directed protein kinase

Rui-Ming Xu, Gilles Carmel1, Robert M.Sweet2, Jeff Kuret1' 3 and Xiaodong Cheng3 W.M.Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, 'MGC Corp., 101 Waukegan Road, Lake Bluff, IL 60044 and 2Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA 3Corresponding authors Communicated by J.D.Watson

The structure of a truncated variant of casein kinase1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate. Key words: casein kinase- 1/nucleotide binding/protein kinase regulation/substrate specificity/structural similarity

Introduction Casein kinase-1 (CK1) is a family of protein kinases common to all eukaryotic cells (Tuazon and Traugh, 1991; Issinger, 1993). These enzymes phosphorylate many proteins in vitro, including SV40 T-antigen (Cegielska and Virshup, 1993), glycogen synthase (Kuret et al., 1985; Roach, 1991), the transcriptional regulators CREM (De Groot et al., 1993) and p53 (Milne et al., 1992). CK1 phosphorylation alters the activity of each of these substrates, and is necessary for full origin unwinding activity of T-antigen. In lower eukaryotes, individual CK1 isoforms perform essential functions in both the cytoplasm and nucleus, where they participate in the regulation of DNA repair pathways and cell morphogenesis (Hoekstra et al., 1991; Robinson et al., 1992; Wang et al., 1992). In addition to its roles in normal cells, CKI has been implicated in the pathogenesis of Alzheimer's disease through the hyperphosphorylation of tau protein, the major component of neurofibrillary tangles (Vincent and Davies, 1993). To carry out its biological function, CKI employs a catalytic domain that differs from most other protein kinases. Initially, on the basis of sequence alignments (Wang et al., 1992), it contains neither the peptide triplet Ala-Pro-Glu in subdomain VIII nor the Arg residue in subdomain XI (Hanks et al., 1988). In other protein kinases, these subdomains interact to form a Glu-Arg © Oxford University Press

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ion pair (Knighton et al., 1991a; De Bondt et al., 1993). Secondly, CK1 isoforms invariably retain high contents of lysines and arginines, resulting in unusually high isoelectric points (pl >9). Thirdly, the substrate selectivity of CK1 appears to be directed toward phosphate groups rather than unmodified amino acids. Evidence for this selectivity comes from studies with synthetic peptide substrates derived from CK1-specific phosphorylation sites in 0-casein (Ser22: Meggio etal., 1991), glycogen synthase (SerlO: Flotow et al., 1990; Flotow and Roach, 1991) and SV40 T-antigen (Serl23: Umphress et al., 1992). Results from all three systems point to the presence of acidic residues N-terminal to a phosphorylatable hydroxyamino acid as the minimal requirement for recognition of synthetic peptides by CK1. For example, the peptide DDDEESITRR is a substrate for CK1 (the underlined serine is the phosphate acceptor), but is phosphorylated with a KM value in the millimolar range (Meggio et al., 1991). Replacement of the aspartates with phosphoserines, however, results in substrates that are phosphorylated with higher kcatlKM values (Meggio et al., 1991; Wang et al., 1993). Phosphoserine has the greatest impact on substrate selectivity when placed at position P-3 (i.e. three residues N-terminal to the phosphorylatable hydroxyamino acid). Thus, CKI isoforms appear to selectively recognize the sequence -S(P)XXS-, where X is any amino acid. To learn more about the unusual structural features of CK1, we crystallized a C-terminal deletion mutant of Cki 1 (Carmel et al., 1994), one of four CKI isoforms in fission yeast (Wang et al., 1994). The truncation, termed CkilA298, eliminates the C-terminal 148 amino acids of Ckil. Although the role of C-terminal domain is not fully understood, part of its function is to target the enzyme to membranes (Wang et al., 1994). The 298-residue catalytic core of Ckil is active catalytically (Carmel et al., 1994), is nearly identical in length to cyclin-dependent kinase 2 (Cdk2) and corresponds to residues 37-319 of cAMPdependent protein kinase (cAPK). Here we report the crystal structure of CkilA298 in complex with MgATP at 2.0 A resolution, and compare it with the structures of other protein kinases: the 2.2 A-resolution model of the ternary complex of mammalian cAPK (pdb code lATP; Zheng et al., 1993) and the 2.4 A-resolution model of the binary complex of human Cdk2 (De Bondt et al., 1993). The resultant CK1 model suggests how the substrate selectivity of this enzyme family is achieved.

Results Quality of the model Refinement statistics for CkilA298 crystallized in a trigonal lattice are summarized in Table I. The current model consists of 288 of 298 possible residues, one complete molecule of ATP, one Mg2+ ion, and 103 water molecules. 1015

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Table I. Crystallographic data and refinement Data collection Space group Unit cell (A)

Number of crystals Total measurements Unique reflections

Rmergea (%)

isomorphous anomalous

Resolution range (A) 20.0-2.71 11.3-3.95 3.95-3.16 3.16-2.76 2.76-2.52 2.52-2.34 2.34-2.20 2.20-2.09 2.09-2.00 Phasing Hg sites (number) Phasing powerb at 3 A Isomorphous Anomalous Figure of merit at 3 A

Refinement Resolution range (A) 11.3-2.00 6.0-2.00 Protein atoms ATP atoms Ordered water molecules Sulfate atoms R.m.s. deviation from ideality Bond lengths Bond angles

P3221 a = b = 78.96 Native

c = 120.38 pCMBS

Dimercurial

8 161782 24245

2 76281 16824

8 66555 15755

8.04 2.35

7.93 5.50

8.66 5.44

93.9 94.3 97.2 93.4 88.4 82.8 74.6 65.1 44.2

92.0

92.2

-

2

2

1.08 2.02

1.37 2.03

Completeness (%)

0.684 Reflections (number) 23860 22976 2386 32 103 10

R-factor (%) 20.7 19.5

0.008 A 1.460

isomorphous data = III- ll X 100, where I is the measured intensity of each reflection, and is the intensity averaged from aR.e,ge ofobservations = >>+ of reflections.

multiple symmetry related Rmerg, of anomalous data 1I1 . :1 1 III i: -E:G WGVPiRTYTHI IRLAD ::k2 :GIPLF KSY'LFQLLQG1AF MSdJ38VLHfvDLK5ZiSEPQNLLXINTCdk2 84 -HDL1A8iT3-\;d....... EGAIKLADPLA FV RTYT

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Fig. 2. Structure-based amino acid sequence alignment of yeast Cki16298, human Cdk2, and mouse cAPK37-319. Identities between pairs of kinases are shown with vertical bars, whereas similarities, grouped as (V, L, I, M), (F, Y, W), (K, R), (E, D), (Q, N), (S, T) and (A, G, P), are indicated by colons. Of CK1 and cAPK, 50 (17%) amino acids are identical and 42 (14%) are similar. Roman numerals mark the beginning of each of the eleven protein kinase subdomains identified by Hanks et al. (1988). Numbering and secondary structure elements are shown for Cki16298. Phosphorylation sites are indicated by the circled letter P. Residues highlighted in Ckil1298 are either invariant (white against black) or similar (shaded) in all known isoforms of CK1 (Keamey et al., 1994). Residues highlighted in cAPK are those identified as being conserved in most protein kinases. Residues highlighted in Cdk2 correspond to the core region with r.m.s. deviation