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• W2100716215 abstract "Article15 July 2003free access The structure of the cell cycle protein Cdc14 reveals a proline-directed protein phosphatase Christopher H. Gray Christopher H. Gray Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Valerie M. Good Valerie M. Good Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Nicholas K. Tonks Nicholas K. Tonks Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author David Barford Corresponding Author David Barford Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Christopher H. Gray Christopher H. Gray Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Valerie M. Good Valerie M. Good Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Nicholas K. Tonks Nicholas K. Tonks Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author David Barford Corresponding Author David Barford Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Author Information Christopher H. Gray1, Valerie M. Good1, Nicholas K. Tonks2 and David Barford 1 1Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB UK 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3524-3535https://doi.org/10.1093/emboj/cdg348 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Cdc14 family of dual-specificity protein phosphatases (DSPs) is conserved within eukaryotes and functions to down-regulate mitotic Cdk activities, promoting cytokinesis and mitotic exit. We have integrated structural and kinetic analyses to define the molecular mechanism of the dephosphorylation reaction catalysed by Cdc14. The structure of Cdc14 illustrates a novel arrangement of two domains, each with a DSP-like fold, arranged in tandem. The C-terminal domain contains the conserved PTP motif of the catalytic site, whereas the N-terminal domain, which shares no sequence similarity with other DSPs, contributes to substrate specificity, and lacks catalytic activity. The catalytic site is located at the base of a pronounced surface channel formed by the interface of the two domains, and regions of both domains interact with the phosphopeptide substrate. Specificity for a pSer-Pro motif is mediated by a hydrophobic pocket that is capable of accommodating the apolar Pro(P+1) residue of the peptide. Our structural and kinetic data support a role for Cdc14 in the preferential dephosphorylation of proteins modified by proline-directed kinases. Introduction In eukaryotes, normal cell cycle progression and viability rely on the dual-specificity protein phosphatase (DSP) Cdc14 (Wan et al., 1992). Organisms with a mutated Cdc14 gene are unable to complete cytokinesis and/or exit from mitosis (Taylor et al., 1997; Morgan, 1999). The Cdc14 proteins of Saccharomyces cerevisiae, Schizosaccharomyces pombe and recently Caenorhabditis elegans have been extensively studied, and two human isoforms (Cdc14A and Cdc14B) were identified on the basis of sequence similarity to the budding yeast protein. Cdc14A and Cdc14B appear to possess similar biochemical properties to their homologues from other species (Bembenek and Yu, 2001; Kaiser et al., 2002). Cdc14 from diverse species share a conserved core of ∼350 amino acids located towards the N-terminus, and which harbours the conserved protein tyrosine phosphatase (PTP) signature motif HC(X)5R(S/T) (Figure 1). Regions C-terminal to the conserved core are highly divergent and share no structural similarities. Figure 1.Structural relationship between eukaryotic Cdc14 proteins. (A) Sequence alignment of budding and fission yeast Cdc14, and human Cdc14A and Cdc14B, within the conserved domain of ∼350 amino acids denoted in blue in (B). Residues that interact with the Pro(P+1) residue of the peptide are indicated by green arrows, residues of the acidic groove by red arrows and critical catalytic site residues by blue arrows. Secondary structural elements in the A- and B-domains are labelled with the suffix A and B, respectively. (B) Schematic of the primary structure of Cdc14 from human and yeast. The conserved domain is shown in blue. Within these regions, human Cdc14B shares 65, 36 and 40% identity with human Cdc14A, S.cerevisiae and S.pombe Cdc14, respectively. The positions of the nucleolar targeting sequence and NES are indicated. Download figure Download PowerPoint Cdc14 of S.cerevisiae is recognized as the ultimate effector molecule of the mitotic exit network (MEN), a signal cascade that promotes the inactivation of the mitotic cyclin-dependent kinase (Cdk) Cdc28 at the end of anaphase (Traverso et al., 2001). The down-regulation of Cdc28 occurs by Cdc14-mediated dephosphorylation of the Cdk-modified residues of Cdh1, a co-activator of the anaphase promoting complex (APC). Activated (dephosphorylated) Cdh1 binds to the APC forming the APCCdh1 complex, the E3-ubiquitin ligase responsible for the ubiquitylation of Clb2 leading to the destruction of the Clb2/Cdc28 complex (Morgan, 1999). Regulation of Cdc14 activity in S.cerevisiae is achieved by three complex mechanisms controlling subcellular localization. For the majority of the cell cycle, Cdc14 is sequestered in the nucleolus by Net1 of the RENT (regulator of nucleolar silencing and telophase) complex (Visintin and Amon, 2000; Traverso et al., 2001). At anaphase, the FEAR (Cdc fourteen early anaphase release) network (Stegmeier et al., 2002) and later the MEN (Jaspersen et al., 1998; Geymonat et al., 2002) promote the release of Cdc14 into the cytoplasm, initially to further regulate its own translocation from the nucleolus, and then to dephosphorylate, hence activating Cdh1, and promote the destruction of Clb2. Inactivation of Cdk activity is further augmented by Cdc14-mediated dephosphorylation of two other Cdk substrates. Dephosphorylation of Sic1 prevents its degradation, hence promoting inhibitory interactions with Cdc28, whereas dephosphorylation of the transcription factor Swi5 stimulates Sic1 gene expression. In contrast to budding yeast, the Cdc14 homologue of S.pombe Clp1 (also termed Flp1) is not required for cyclin degradation or the activation of the APC, and thus does not appear to promote mitotic exit (Cueille et al., 2001). However, Clp1 does interact with the fission yeast homologues of the MEN that are termed the SIN (septation initiation network). This network co-ordinates cytokinesis during nuclear division, and Clp1 localizes to both the mitotic spindle and the contractile ring. Clp1 differs from S.cerevisiae Cdc14 by regulating the G2/M transition. Cells deleted for Clp1 enter mitosis prematurely, whereas overexpression of the phosphatase delays mitotic entry by preventing dephosphorylation of Cdc2 on Tyr15 (Trautmann et al., 2001). Interactions with the cytoskeleton to facilitate cytokinesis also apply to the recently characterized Cdc14 of C.elegans, CeCDC14, which is essential for the localization of key components to the central spindle in anaphase and the midbody in telophase. Depletion of CeCDC14 by RNAi in embryos resulted in lethality as a consequence of poor central spindle formation and cytokinesis, rather than a defect in mitosis or the nuclear cycle. It is proposed that successful central spindle formation is dependent on CeCDC14 dephosphorylation of one or several Cdk-modified substrates (Gruneberg et al., 2002). The roles of the two human isoforms of Cdc14 are poorly understood, but the potential for the regulation of both mitotic exit and cytokinesis is likely. In situ observations of fluorescently labelled Cdc14A in human cells show a localization and recruitment to interphase centrosomes following nuclear export. This release is mediated by a nuclear export signal (NES) in Cdc14A, located just beyond the C-terminal reaches of the phosphatase domain. Mutation of the NES results in the retention of Cdc14A in the nucleolus. Conversely, the N-terminal 44 amino acids are responsible for localizing Cdc14B to the nucleolus throughout interphase (Kaiser et al., 2002) (Figure 1B). Overexpression of Cdc14A results in premature centrosome splitting and an excessive number of mitotic spindles, whereas down-regulating endogenous Cdc14A using siRNA impairs centrosome splitting and prevents successful cytokinesis (Mailand et al., 2002). While the range of substrates for Cdc14A or Cdc14B is not well defined, in vitro studies have shown that Cdc14A has a clear preference for substrates of proline-directed kinases (Cdks and MAP kinases) modified at pSer/pThr-Pro motifs (Kaiser et al., 2002; Trautmann and McCollum, 2002). This is consistent with the observation that, just as the yeast Cdc14 dephosphorylates a Cdk-modified Cdh1, human Cdc14A can activate the human Cdh1 to constitute the APCCdh1 in vitro (Bembenek and Yu, 2001). Cdc14B may play an important role within the nucleolus during interphase and, as the nuclear membrane dissolves at prophase, Cdc14B will be released into the cytoplasm and may act on a wider range of Cdk- or MAPK-modified substrates during mitosis. The diverse roles of Cdc14 to regulate cytokinesis and mitosis suggest that Cdc14 will act on different targets in each case to effect a subtly different result. However, the underlying element of continuity is the dephosphorylation of Cdk-modified substrates (Visintin et al., 1998; Trautmann and McCollum, 2002). A comprehensive understanding of the mechanisms of catalysis, and specificity for Cdk-modified substrates by Cdc14, requires structural investigation. To address this question, we have determined crystal structures of the core domain of human Cdc14B in both the apo state, and as a complex with a phosphopeptide substrate, at 2.2 Å resolution. These are the first reported X-ray crystallographic data for Cdc14. The overall structure illustrates a novel fold of two DSP domains arranged in tandem that may have evolved from an early gene duplication event of an ancestral DSP gene. The structure of Cdc14B demonstrates the molecular basis of its specificity for substrates with pSer-Pro and pThr-Pro motifs that are common to Cdk- and MAP kinase-modified proteins. Results Structure determination To understand the three-dimensional (3D) structure of human Cdc14B (Mr 53 kDa), we expressed the full-length protein using the insect cell/baculovirus system, and purified the protein to near homogeneity. This form of the protein did not readily crystallize, although the appearance of small Cdc14B crystals were noted in hanging drops from an individual preparation of the protein after a period of 3 months. Analysis of the protein mass in the protein/crystal drop using SDS–PAGE revealed spontaneous and partial degradation of Cdc14B to a size of ∼40 kDa, suggesting that the crystals grew from a truncated form of the protein. Elective limited proteolysis was used to delineate the structurally stable domain that corresponded to the spontaneously truncated protein. Limited proteolysis of full-length Cdc14B using three different proteases yielded a stable product of ∼40 kDa, similar in size to the truncated form of Cdc14B obtained by spontaneous degradation. Edman sequencing revealed the N-terminus as Pro44, whereas an estimation of the C-terminus was based on the C-terminal boundary of the conserved catalytic domains of Cdc14A, Cdc14B and S.cerevisiae Cdc14. The resultant protein (residues Pro44–His386) when purified had a molecular mass, as judged by SDS–PAGE, equivalent to the partially degraded Cdc14B obtained by limited trypsinolysis and, moreover, readily crystallized. Significantly, this region of Cdc14B corresponds to the segment of sequence conservation within Cdc14 sequences from diverse species, and therefore represents the Cdc14 catalytic core (Figure 1). Determination of the structure of wild-type apo Cdc14B was performed using the single anomalous dispersion method utilizing tungstate, a phosphate mimic and catalytic site inhibitor, as a heavy atom derivative. The concentration of tungstate used to derivatize Cdc14B was estimated from the concentration required to inhibit the Cdc14 catalytic activity towards p-nitrophenol-phosphate (pNPP; data not shown). The structure of wild-type apo Cdc14B was solved to 2.5 Å resolution, the diffraction limit of these crystals. Subsequently, we obtained crystals of a Cdc14B–phosphopeptide complex by substituting serine for the catalytic Cys314 residue. These crystals diffracted to 2.2 Å and were solved by molecular replacement using the apo Cdc14B structure (Table I). In both structures, residues Pro44–Lys379 are well defined in the electron density maps, whereas the C-terminal seven residues are disordered. Apo and complex Cdc14B share virtually identical conformations (see below). Because the higher resolution of the Cdc14–peptide complex resulted in a better model for the protein, we use this form as the basis of the description of molecular structure. Table 1. Crystallographic data and refinement statistics Crystal parameters Native Tungstate Peptide Space group a (Å) 114.82 114.74 114.76 b (Å) 52.08 51.93 53.15 c (Å) 65.19 64.96 64.17 β (°) 118.24 118.63 117.48 Z 1 1 1 Data collection and processing statistics Resolution (Å) 2.5 2.6 2.2 Measurements (n) 38 931 30 373 62 805 Unique (n) 11 713 10 480 17 049 % complete 99.2 (97.1) 99.5 (99.6) 97.2 (92.2) Rsyma 0.106 (0.345) 0.119 (0.331) 0.073 (0.24) I/σI 6.2 6.8 7.1 (2.9) Refinement statistics Resolution (Å) 2.5 2.6 2.2 No. of reflectionswork (%) 10 814 (91.6) 9931 (84.5) 16 051 (91.1) No. of reflectionsfree (%) 589 (5.0) 553 (4.7) 997 (5.7) Atoms (protein and ligands) 2744 2748 2771 Water molecules 57 24 143 Rfreeb 0.282 0.280 0.254 Rfactorc 0.204 0.210 0.208 R.m.s.d. bond lengths (Å) 0.011 0.0079 0.013 R.m.s.d. bond angles (°) 1.55 1.30 1.53 Cdc14 is composed of two structurally equivalent domains The molecular architecture of Cdc14B is composed of two similar sized domains arranged in tandem, associated via an extensive interface to form a single globular whole (Figure 2). Strikingly, both domains adopt a DSP-like fold. A linker α-helix (residues 199–212) connects the two domains. The conserved PTP signature motif (Cys-[X]5-Arg) that defines the catalytic centre of all PTP-family members is located within the C-terminal domain (B-domain, residues 213–379) and, together with the location of the phosphopeptide substrate in the catalytically inactive C314S mutant, identified the position of the catalytic site of Cdc14. As expected, tungstate bound to this site. Although the centre of the catalytic site is formed from B-domain, two loops from the N-terminal domain (A-domain) also contribute to the catalytic site, facilitating peptide substrate specificity (see below). Figure 2.Ribbon diagram of Cdc14B. Two orthogonal views showing the overall structure of the Cdc14–phosphopeptide complex. The A- and B-domains are green and cyan, respectively, and the inter-domain α-helix is yellow. There is a large solvent-accessible surface area of 2108 Å2 buried between the two domains. The phosphopeptide substrate is shown as a red coil, and key catalytic site loops are labelled. Figures were made with PyMOL (http://www.pymol.org). Download figure Download PowerPoint The conformation of apo wild-type Cdc14B is virtually identical to both the Cdc14B–tungstate complex and the Cdc14B–phosphopeptide complex. Equivalent Cα atoms of apo Cdc14B and the Cdc14–peptide complex superimpose within an r.m.s.d. of 0.46 Å, and there is no indication of relative domain movements on association of peptide. The structure of apo Cdc14B that we describe here is the first example of a DSP crystallized in the absence of an oxy-anion bound to the catalytic site. Significantly, the conformation of the invariant WPD (Trp-Pro-Asp) loop, connecting β4 and α3, which bears the essential and invariant general acid/base Asp287 residue, adopts the closed, catalytically competent conformation in both apo and complex states. This finding demonstrates, that for Cdc14, in contrast to all known tyrosine specific PTPs, the binding of substrate is not required to induce closure of the WPD loop (Jia et al., 1995). The B-domain contains the catalytic centre and is structurally related to PTEN The architecture of the B-domain is highly reminiscent of other DSPs (Figures 2 and 3) (Barford et al., 1998). These proteins share the general characteristic of having a central mainly parallel β-sheet of five strands, with two α-helices on one side of the sheet. The fifth and middle β-strand leads into the conserved PTP signature motif that forms the base of the catalytic site, which in turn is connected to one of four α-helices that pack onto the opposite side of the β-sheet. A search of the protein database (PDB; Berman et al., 2000) using the DALI server (Holm and Sander, 1996) revealed that surprisingly the B-domain of Cdc14 is most similar to the phosphoinositol 3,4,5 tris-phosphate (PIP3) phosphatase PTEN (Lee et al., 1999) (Figure 3A), and the phosphatase domain of the mRNA capping enzyme (Changela et al., 2001) (Table II). A structural feature critical for the ability of PTEN to dephosphorylate the D3 position of its negatively charged PIP3 substrate are two conserved lysine residues within the PTP motif: (HCKAGKGR; lysines in bold) and a His residue in the WPD loop (Lee et al., 1999). Interestingly, the PTP motif of Cdc14 (HCKAGLGR) is also reminiscent of PTEN, although the His residue of the WPD loop of PTEN is a glycine (Gly288) in Cdc14, and therefore it is unlikely that Cdc14 functions to dephosphorylate lipid substrates. The most closely related protein phosphatases to Cdc14 are kinase-associated phosphatase (KAP) (Song et al., 2001) and vaccinia H1-related phosphatase (VHR) (Yuvaniyama et al., 1996) (Table II). Figure 3.Structural relatedness of the A- and B-domains of Cdc14B. (A) Comparison of structures of the A- and B-domains of Cdc14B and the phosphatase domain of PTEN. In the upper panel, the three domains are shown in the same orientation, and a stereoview of the A-domain (green) and B-domain (blue) superimposed is shown in the lower panel. (B) Structure-based sequence alignment of domains A and B of Cdc14B. Equivalent secondary structural elements are suffixed with ‘A’ and ‘B’ for domains A and B, respectively. Download figure Download PowerPoint Table 2. Highest structural relationships between domains of Cdc14B and protein structures deposited at the PDB as defined by DALI Protein 1 Protein 2 No. of aligned residues R.m.s.d. of Cα (Å) Z-scorea A-domain PTEN 126 3.0 9.9 VHR 124 3.0 9.8 B-domain 119 2.6 9.6 Cdc14B mRNA capping 121 2.7 9.5 protein KAP 123 3.0 9.0 B-domain PTEN 150 2.0 19.5 mRNA capping 150 2.2 18.7 protein KAP 153 2.7 18.2 VHR 141 2.4 16.6 PTP1B 151 2.5 13.7 aZ-scores above two are significant (Holm and Sander, 1996). The A-domain has a DSP-like fold The 3D architecture of the A-domain (residues 44–198) bears a remarkable resemblance to the B-domain of Cdc14. As shown in Figure 3A, the secondary structural elements of the A-domain superimpose closely onto the conserved core elements of the B-domain, and the two domains share the same secondary structure topology and polypeptide connectivities. Overall, the Cα atoms of 119 equivalent residues superimpose within an r.m.s.d. of 2.6 Å and the Z-score, a measure of the structural similarity in standard deviations above the expected value between two molecules, is 9.6 (Table II). Interestingly, this analysis indicated that the PTP/DSP family is structurally unique, such that a similar topology does not occur in other proteins. These findings suggest that the A-domain of Cdc14 resulted from divergent evolution from an ancestral PTP/DSP family member, possibly from a gene duplication event of the existing catalytically active B-domain. The structural similarity between the A- and B-domains of Cdc14B is not reflected in any sequence similarity. A structure-based alignment of the A- and B-domains indicates only 11% sequence identity (Figure 3B). Importantly, none of the catalytic site residues, including the catalytic site Cys and Arg residues, characteristic of PTP/DSPs, is present in the A-domain. Significantly, the structure of the A-domain suggests that it would be unable to bind phosphate in the equivalent region of the molecule to the phosphate-binding cradle formed by the PTP signature motif of the B-domain. In the A-domain, an insertion of two residues at the N-terminus of α4A, equivalent to the α4B helix which forms the base of the catalytic site in the B-domain (Figure 3B), alters the conformation of the A-domain so that it no longer forms a phosphate-binding cradle. Consistent with the notion that the A-domain is incapable of binding a phosphate moiety, we observed tungstate at 25 mM bound only to the catalytic site of the B-domain. Other variations between the A- and B-domains include a 13 residue insertion in the α5A/α6A loop, which contributes to the peptide-binding groove, and the counterpart to the WPD loop of the B-domain is four residues longer in the A-domain (Figure 3B). Finally, there are no equivalents of the α1 and α2 helices, and β4 strand, conserved in the B-domain of Cdc14B and other DSPs. The peptide-binding groove is selective for proline-directed peptides A unique feature of the catalytic site of Cdc14B is its location within a long groove (25 Å long and 10 Å wide), at the interface of the A- and B-domains. Residues of two loops of the A-domain, the extended WPD(A) and α5A/α6A loops, create one side of the groove (Figures 2, 4 and 5A). The WPD and Q-loops of the B-domain form the opposite face of the channel, whereas the inter-domain linker α-helix is positioned at the entrance to one end of the channel. Significantly, this region of the linker α-helix is rich in acidic residues (Glu206, Glu209 and Asp215) that cluster to generate a pronounced acidic groove leading to the catalytic site (Figure 5A). Cdc14 is genetically and biochemically linked to the dephosphorylation of Cdk substrates (Visintin et al., 1998; Kaiser et al., 2002), suggesting that the phosphatase must be capable of dephosphorylating phosphoserine/threonine residues located immediately N-terminal to a proline residue. Moreover, because Arg and Lys residues are usually located at the P+2 and P+3 positions C-terminal to Cdk sites of phosphorylation (Songyang et al., 1994; Holmes and Solomon, 1996; Kreegipuu et al., 1999), it is likely that Cdc14 will display some selection for phosphopeptides with basic residues C-terminal to the phospho-amino acid. It is, therefore, tempting to suggest that the cluster of acidic residues at the catalytic groove of Cdc14 may function to confer this selectivity. Figure 4.Catalytic site of the Cdc14B–phosphopeptide complex. (A) Stereoview of 2Fo − Fc (cyan) and Fo − Fc (red) stimulated annealing omit electron density maps of the phosphopeptide, contoured at 1σ and 3σ, respectively. (B) Stereoview showing details of the catalytic site–peptide interactions, indicating the hydrophobic pocket that accommodates the Pro(P+1) residue. Hydrogen bonds are shown as dotted grey lines, and the van der Waals contacts between the peptide Pro(P+1) residue and Cdc14B are shown as yellow dotted lines. (C) Expanded view of the catalytic site with atoms coloured according to atomic B-factors with colours ramped from blue to red to denote B-factors ranging from to 13 to 52 Å2. Download figure Download PowerPoint Figure 5.Molecular surface of the Cdc14 catalytic channel with bound phosphopeptide substrate. (A) Electrostatic potential of Cdc14 catalytic channel, negative and positive electrostatic potentials are shown as red and blue, respectively. (B) Surface plot showing conservation of residues at the catalytic site of Cdc14 based on a sequence alignment of budding and fission yeast Cdc14 and human Cdc14A and Cdc14B. The colours are ramped from red to blue to denote invariant to unconserved residues. Figures drawn using SHARP (Nicholls et al., 1991). (C) Comparison of Cdc14–phosphopeptide complex (green) and PTP1B–IRK complex (purple) showing the PTP, WPD and Q-loops and phosphopeptide substrates of both proteins super imposed. The pTyr recognition loop is unique to PTP1B, and Tyr46 of the pTyr loop and Phe182 of the WPD loop of PTP1B create the selectivity for pTyr residues. A shallow catalytic channel in Cdc14 for pSer/Thr peptides is possible because of a Gly in the WPD loop in place of Phe and the absence of the pTyr loop. Download figure Download PowerPoint To address the basis of Cdc14–substrate recognition, we co-crystallized a catalytically inactive Cys314 to Ser mutant of Cdc14 with a phosphopeptide of sequence A-pS-P-R-R-R, comprising the generic features of a Cdk substrate: a proline at the P+1 position and basic residues at P+2 to P+4. The structure of the Cdc14–phosphopeptide complex is shown in Figures 2, 4 and 5. Only the three residues A-pS-P are clearly delineated in electron density omit maps (Figure 4A). Density corresponding to the C-terminal basic residues is not visible, suggesting that these amino acids adopt multiple conformations when bound to Cdc14B. Atomic temperature factors of the peptide are in the same range as surface residues of the enzyme (Figure 4C). In the Cdc14–phosphopeptide complex, the Pro residue of the peptide is clearly defined as being in the trans isomer. With this conformation, residues C-terminal to the pSer-Pro motif will be directed into the acidic groove at the catalytic site and, importantly, a peptide with a cis proline would be unable to engage with the catalytic site due to a steric clash with the sides of the groove. This finding suggests that the pSer/pThr-Pro specific cis–trans peptidyl prolyl isomerase Pin1 may function to facilitate Cdc14 activity (Lu et al., 2002). Interactions of the substrate phosphoserine residue with the catalytic site are reminiscent of phospho-amino acids bound to other protein phosphatases (Jia et al., 1995; Salmeen et al., 2000; Song et al., 2001); its phosphate moiety is coordinated by residues of the PTP loop, positioning it adjacent to the nucleophilic thiol group of Cys314 (Figures 4B and 5C). Similarly to PTP1B, the carboxylate group of the general acid Asp287 (Asp181 of PTP1B) is placed to donate a hydrogen bond to the Oγ atom of the pSer substrate. Interestingly, the peptide orientation is opposite to that of peptides bound to the phosphotyrosine-specific PTP1B. In PTP1B, Asp48 of the pTyr recognition loop forms bidendate interactions to the amide nitrogen atoms of the pTyr and P+1 residues, helping to define the substrate peptide orientation (Jia et al., 1995; Salmeen et al., 2000). There is no equivalent to the pTyr recognition loop of pTyr-specific PTPs in Cdc14, and interactions between main-chain groups of the peptide and the catalytic site do not appear to be a significant feature of the Cdc14–phosphopeptide complex. Moreover, residues of PTP1B that define the depth of the catalytic site pocket, and hence the selection for pTyr substrates, i.e. Tyr46 of the pTyr recognition loop and Phe182 of the WPD loop, are absent from Cdc14 (Figure 5C). Apart from interactions involving the phospho-amino acid residue, the only other significant contacts formed between the phosphatase and peptide are contributed by the P+1 proline residue. The prolyl ring of Pro (P+1) is located in a hydrophobic pocket created by the side chains of Phe85 and Tyr170 of the A-domain WPD(A) and α5A/α6A loops, respectively, and two B-domain residues: Leu318 of the PTP loop and Ile353 of the Q-loop (Figures 4 and 5). Significantly, the aliphatic side chains of Phe85, Leu318 and Ile353 form optimal van der Waals contacts with the Pro(P+1) prolyl ring (Figure 4B). The location of this hydrophobic pocket immediately adjacent to the catalytic site for the phospho-amino acid confers the selectivity of Cdc14 for peptides with proline residues at the P+1 position. The tertiary nitrogen of the proline amide has no hydrogen-bonding potential, allowing it to be accommodated within an apolar environment. In contrast, the requirement to satisfy the hydrogen-bonding potential of the amide nitrogen of all other amino acids would select against peptides with non-proline residues at this position. Interestingly, a similar non-polar pocket at the P+1 position of the substrate peptide-binding site in Cdk2 determines its Pro-directed substrate selectivity (Brown et al., 1999). The conservation of residues that define the P+1 pocket in Cdc14 genes from diverse species suggests a common mechanism by which Cdc14 phosphatases are directed towards proline-containing peptides (Figures 1 and 5B). Finally, in the Cdc14–peptide complex, the side chain of Ala(P-1) is directed" @default.
• W2100716215 created "2016-06-24" @default.
• W2100716215 creator A5042719296 @default.
• W2100716215 date "2003-07-15" @default.
• W2100716215 modified "2023-10-04" @default.
• W2100716215 title "The structure of the cell cycle protein Cdc14 reveals a proline-directed protein phosphatase" @default.
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• W2100716215 doi "https://doi.org/10.1093/emboj/cdg348" @default.
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