Báo cáo khoa học: High levels of structural disorder in scaffold proteins as exemplified by a novel neuronal protein, CASK-interactive protein1 pot

High levels of structural disorder in scaffold proteins as exemplified by a novel neuronal protein, CASK-interactive protein1 ´ ´ ´ ´ ´ Annamaria Balazs1,*, Veronika Csizmok2,*, Laszlo Buday1,2, Marianna Rakacs2, Robert Kiss3, ´ ´ ´ Monika Bokor4, Roopesh Udupa2, Kalman Tompa4 and Peter Tompa2 Department of Medical Chemistry, Semmelweis University Medical School, Budapest, Hungary Biological Research Center, Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary ´ Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eotvos Lorand University, Budapest, Hungary ă ă Research Institute for Solid State Physics and Optics, Hungarian Academy of Sciences, Budapest, Hungary Keywords anchor; docking; post-synaptic density; scaffold; unstructured Correspondence P Tompa, Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina ut 29, 1113 Budapest, Hungary Fax: +36 466 5465 Tel: +36 279 3143 E-mail: tompa@enzim.hu *These authors contributed equally to this work (Received 26 February 2009, revised 15 April 2009, accepted 12 May 2009) doi:10.1111/j.1742-4658.2009.07090.x CASK-interactive protein1 is a newly recognized post-synaptic density protein in mammalian neurons Although its N-terminal region contains several well-known functional domains, its entire C-terminal proline-rich region of 800 amino acids lacks detectable sequence homology to any previously characterized protein We used multiple techniques for the structural characterization of this region and its three fragments By bioinformatics predictions, CD spectroscopy, wide-line and 1H-NMR spectroscopy, limited proteolysis and gel filtration chromatography, we provided evidence that the entire proline-rich region of CASK-interactive protein1 is intrinsically disordered We also showed that the proline-rich region is biochemically functional, as it interacts with the adaptor protein Abl-interactor-2 To extend the finding of a high level of disorder in this scaffold protein, we collected 74 scaffold proteins (also including proteins denoted as anchor and docking), and predicted their disorder by three different algorithms We found that a very high fraction (53.6% on average) of the residues fall into local disorder and their ordered domains are connected by linker regions which are mostly disordered (64.5% on average) Because of this high frequency of disorder, the usual design of scaffold proteins of short globular domains (86 amino acids on average) connected by longer linker regions (140 amino acids on average) and the noted binding functions of these regions in both CASK-interactive protein1 and the other proteins studied, we suggest that structurally disordered regions prevail and play key recognition roles in scaffold proteins Structured digital abstract l MINT-7034649: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with L1CAM (uniprotkb:P32004) by two hybrid (MI:0018) l MINT-7034677: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with NCK1 (uniprotkb:P16333) by two hybrid (MI:0018) l MINT-7034706: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Stathmin-3 (uniprotkb:Q9NZ72) by two hybrid (MI:0018) Abbreviations Abi2, Abl-interactor-2; Caskin1, CASK-interactive protein1; CBP, CREB-binding protein; GFP, green fluorescent protein; GST, glutathione transferase; IDP, intrinsically disordered protein; IUP, intrinsically unstructured protein; MAPK, mitogen-activated protein kinase; PRD, proline-rich region; PSD, post-synaptic density; SAM, sterile a motif; Ste5, Sterile 3744 FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS V Csizmok et al High levels of disorder in scaffold proteins l l l l l l l l l MINT-7034579: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with ABI2 (uniprotkb:Q9NYB9) by two hybrid (MI:0018) MINT-7034720: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Synaptotagmin (uniprotkb:P21579) by two hybrid (MI:0018) MINT-7034691: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Neurexin-2 (uniprotkb:Q9P2S2) by two hybrid (MI:0018) MINT-7034617: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with CASK (uniprotkb:P07498) by two hybrid (MI:0018) MINT-7034748: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with SIAH1 (uniprotkb:Q8IUQ4) by two hybrid (MI:0018) MINT-7034663: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Myosin-Ib (uniprotkb:O43795) by two hybrid (MI:0018) MINT-7034734: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Septin-4 (uniprotkb:O43236) by two hybrid (MI:0018) MINT-7034634: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with EPHA2 (uniprotkb:P29317) by two hybrid (MI:0018) MINT-7034765, MINT-7034783: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with ABI2 (uniprotkb:Q9NYB9) by pull down (MI:0096) Introduction Recently, novel scaffold proteins have been discovered in the brain, particularly in neuronal cells, and are referred to as the CASK-interactive protein (Caskin) family [1] Caskin1 and its isoform Caskin2, which are present in the post-synaptic density (PSD), are multidomain proteins possessing six ankyrin repeats, two sterile a motifs (SAM domains) and a single SH3 domain in the N-terminal part (cf Fig 1) In contrast, there are no recognizable domains in the C-terminal part, which is dominated by a long proline-rich region [1], designated as the proline-rich domain (PRD) in this work Caskin1 can bind the Cask adaptor protein [1], Abl-interactor-2 (Abi2), and another nine proteins shown in this work, and is presumably involved in the signal pathway related to the Abl tyrosine kinases (A Balazs, V Csizmok, P Tompa, R Udupa & L Buday, unpublished results) The molecular mechanism of the function of Caskins is not known at Fig The diagram at the bottom shows a schematic representation of the domain structure of Caskin1 The N-terminal half contains six ankyrin repeats, one SH3 domain and the two SAM domains, whereas the C-terminal half contains no recognizable domain, and has been designated as a proline-rich region ⁄ domain (PRD) The proline-rich region was cut into three parts (PRD1, PRD2 and PRD3), cloned and characterized individually in this work Above the scheme is the prediction by the IUPred algorithm, which shows that the entire PRD region is probably intrinsically disordered (the score is above 0.5) FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS 3745 High levels of disorder in scaffold proteins V Csizmok et al present As a result of their large size, the capacity to bind multiple partners and the lack of catalytic domains, they probably fall into the class of scaffold proteins, which bind components of a signal transduction pathway simultaneously and ensure the specificity and efficiency of signal propagation [1] As the long regions of these proteins often lack any sequence similarity to other proteins and appear to lack folded structural domains, we anticipated that structural disorder may be a general feature of scaffold proteins In a recent review, structural disorder in several scaffold proteins and in other proteins of multiple binding partners (without adherence to the accepted definition of scaffolds) has been suggested and analysed [2] As a result of the rapid advance of knowledge on intrinsically disordered ⁄ unstructured proteins (IDPs ⁄ IUPs), the concept of protein disorder has gained general recognition recently [3–6] Physical evidence exists for the disorder of about 500 proteins [7], and bioinformatics predictions suggest that disorder is prevalent in the proteome of eukaryotes, with more than 10% of their proteins being fully disordered [8–10] Disorder is most often implicated in signalling and regulatory functions, and its functional benefits often manifest themselves in protein–protein recognition [5,11] One advantage often referred to is that their extended structure enables IDPs to have a large interaction capacity with small protein size [12], which might be directly related to the involvement of disorder in scaffold proteins In fact, there is an elevated level of disorder in hub proteins, i.e proteins involved in multiple interactions [13–16], and disorder increases with the number of proteins in multiprotein complexes [17] The functional role of structural disorder has been noted in a few scaffold proteins, such as Sterile (Ste5) [18], BRCA1 [19], CREB-binding protein (CBP) [4] and Mypt1 [20], and it has been suggested that flexibility provided by disorder is instrumental in overcoming steric hindrance in the assembly of large multiprotein complexes [21] Motivated by the apparent relationship between protein disorder and scaffold function, in this article, we provide evidence that the proline-rich region of the newly recognized Caskin1 is intrinsically disordered To extend this finding, we also collected 74 scaffold proteins (also including proteins denoted as anchor and docking) and examined them by three different disorder predictor algorithms, i.e IUPred [22,23], VSL2 [24,25], and FoldIndex [26] We found that, in these proteins, the frequency of disorder is very high (49.7%, 63.36% and 47.82% predicted by IUPred, VSL2 and FoldIndex, respectively), which is similar to 3746 that in the most disordered functional class, RNA chaperones [27] The implications of these findings with respect to the function of Caskin1, and of scaffold proteins in general, are discussed Results Structural characterization of Caskin1 fragments As described in the introductory paragraphs, the N-terminal half of Caskin1 contains a number of well-known domains involved in protein–protein interaction, such as the ankyrin repeats, SH3 and SAM domains (Fig 1) The three-dimensional structures of these domains have been well characterized [28–30] However, the C-terminal part of Caskin1 does not contain any domain, but possesses several proline-rich stretches Because proline is incompatible with repetitive secondary structural elements [31] and is known to be enriched in IDPs [5], we assumed that the C-terminus of Caskin1 might be intrinsically disordered This expectation was first confirmed by bioinformatics predictions by the IUPred algorithm (Fig 1) High IUPred scores indicate that the entire proline-rich region of Caskin1 (amino acids 603–1430) is disordered To confirm this prediction, a variety of experimental approaches were also applied, as earlier it has been suggested [5] that, as a result of the limitations of most techniques, a multitude of approaches need to be applied for the conclusive demonstration of disorder The full-length proline-rich region of Caskin1 with a histidine tag on its C-terminus (PRD-His) was cloned and expressed in bacteria However, the expression of this construct was rather difficult because of the high proteolytic sensitivity of the protein, characteristic of IDPs Therefore, only CD, gel filtration and limited proteolysis experiments could be performed, which not require large amounts of protein For detailed studies, the full-length proline-rich region was cut into three parts, selected for splitting at sites of high local disorder in the IUPred prediction (PRD1-His, Lys603– Lys804; PRD2-His, Val805–Ala1199; PRD3-His, Glu1200–Glu1430), cloned into PQE2 and pET20b vectors with a C-terminal His tag and expressed in Escherichia coli One important feature of IDPs is their heat stability Therefore, purification of the full-length proline-rich region and its fragments from the bacterial extracts was started by boiling the proteins at 100 °C for and loading the supernatants on an Ni–agarose affinity chromatograph The heat stability of the fragments and of full-length PRD-His during purification FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS V Csizmok et al provides the first line of experimental evidence for disorder The CD spectrum of PRD-His shows a minimum at 202 nm (Fig 2A), which is characteristic of a protein in a largely disordered conformation The CD spectra of the separate PRDs also show characteristic minima around 200 nm (Fig 3A), which underscores the unstructured nature of these regions In the case of PRD2-His, and a little less in the case of PRD3-His, a small shoulder at around 220 nm appears, which indicates secondary structural elements in this region of the protein In addition, the sum of the spectra of the three fragments almost completely reproduces the spectrum of full-length PRD-His (Fig 3B), which confirms the overall random structure of the proline-rich region, i.e the lack of discernible long-range interactions in this region of Caskin1 Another characteristic feature of IDPs is their extreme sensitivity to proteolysis [5] At typical protease concentrations at which globular proteins are hardly affected, these proteins are degraded rapidly and completely In accordance with this, PRD-His shows a greater sensitivity to proteolysis with a protease of wide substrate specificity, subtilisin, than does the globular control protein BSA (Fig 2B); this provides an indication of its disordered conformation Gel filtration data also verify the disordered nature of the proline-rich region, as the apparent molecular mass (mapp) of PRD-His (334.5 kDa) is 3.9 times higher than the real value (85.9 kDa) (Fig 2C) The three fragments also show a high apparent molecular mass: 4.5 (PRD1-His, 95.5 kDa), 2.2 (PRD2-His, 91.9 kDa) and 5.4 (PRD3-His, 125.4 kDa) times higher than the real molecular mass (21, 41.7 and 23.2 kDa, respectively) (Fig 3D) Because the column was calibrated with globular proteins, these ratios suggest a largely unfolded conformational state, as values of mapp ⁄ m = 4–5 are typical of fully disordered proteins [20] We have demonstrated previously that the high hydration potential of IDPs can be detected by wideline 1H-NMR measurements [32,33] This technique is suitable for the measurement of the amount of bound water after freezing out bulk water We compared the temperature dependence of the mobile water fractions of the three fragments PRD1-His, PRD2-His and PRD3-His (Fig 3C) The amount of water in the hydrate layer far exceeds that of BSA and approaches that of ERD10, an IDP characterized previously [33], which provides further evidence for the open and largely solvent-exposed nature It is of note that the mobile water fraction of PRD2-His shows some deviation from that of the other two fragments, i.e the level High levels of disorder in scaffold proteins Fig Structural characterization of the proline-rich region of Caskin1 (PRD-His) (A) CD spectrum of PRD-His; the large minimum at 202 nm is typical of IDPs (B) Limited proteolysis experiment with a broad substrate specificity enzyme, subtilisin, at : 2000 enzyme to substrate ratio Aliquots were withdrawn at times s, 10 s, 30 s and min, and run on SDS-PAGE Caskin1 is much more sensitive to the enzyme than is the control globular protein BSA (C) Gel filtration chromatography of control globular proteins ( , see Materials and methods) and PRD-His of Caskin1 (h) PRD is an extended, random coil-like protein, with an mapp value 3.9 times that of its real m value of hydration of this fragment is lower than that of the other two, which indicates some local preference for ordering within this region FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS 3747 High levels of disorder in scaffold proteins V Csizmok et al A B C D Fig Structural characterization of fragments of PRD (A) Far-UV CD spectra of PRD1-His (blue), PRD2-His (green) and PRD3-His (red) All spectra show a characteristic minimum at around 200 nm, which underscores the unstructured nature of the proline-rich region (B) Comparison of the far-UV CD spectrum of the full-length PRD-His (full line) and the sum of the spectra of PRD1-His, PRD2-His and PRD3-His (broken line) The sum of the spectra of the three fragments reproduces the spectrum of PRD-His, which confirms the overall random structure of the full-length proline-rich region and the lack of appreciable long-range structural organization within this region of the protein (C) The temperature dependence of the mobile water fraction of PRD1-His (blue), PRD2-His (green) and PRD3-His (red), compared with that of the globular control BSA (cyan) and the disordered control ERD10 (black) The large amount of water in the hydrate layer of PRDs suggests their open, solvent-exposed conformations (D) Gel filtration chromatography of the fragments PRD1-His, PRD2-His and PRD3-His shows that all three fragments have an extended conformation with mapp values 4.5, 2.2 and 5.4 times higher than the real m values, respectively The one-dimensional 1H-NMR spectra of the PRDHis fragments (Fig 4) also underscores a largely disordered conformational state Chemical shifts show a poor dispersion, i.e amide proton signals are clustered within a half-p.p.m range centred at p.p.m., whereas the methyl group protons are clustered at around p.p.m Such a limited dispersion and signal overlap in 1H chemical shifts are typical of IDPs [34] The proline-rich regions of Caskin1 interact with Abi2 To demonstrate that the proline-rich regions characterized above are biochemically functional, we studied the 3748 interaction of Caskin1 fragments with Abi2, which is an adaptor protein identified originally by its interaction with Abl tyrosine kinase [35] Caskin1 was cut into five regions and expressed as glutathione transferase (GST) fusion proteins These protein regions represent the ankyrin repeats and the SH3 domain together (ANK ⁄ SH3-GST), the two SAM domains (SAM-GST) and the three proline-rich regions (PRD1–3-GST) of the C-terminal PRD The full-length PRD of Caskin1 was also expressed (PRD-GST) Green fluorescent protein (GFP)-tagged Abi2 was expressed in COS7 cells, extracts of which were used for the GST pull-down assay As shown in Fig 5, the first and second prolinerich regions of Caskin1 (PRD1-GST and PRD2-GST) FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS V Csizmok et al A High levels of disorder in scaffold proteins the full-length PRD-GST has binding characteristics similar to that of PRD2-GST These in vitro data suggest that the proline-rich fragments of C-terminal Caskin1 are functional and may interact with SH3 domain-containing proteins, such as Abi2 [We have also found an in vivo association and colocalization of Abi2 with Caskin1 (A Balazs, V Csizmok, P Tompa, R Udupa & L Buday, unpublished results).] Caskin1 is a scaffold protein B Fig (A,B) One-dimensional 1H-NMR spectrum of PRD3-His shows a narrow p.p.m range and limited dispersion, typical for an unfolded polypeptide (B is the enlarged part of A between 6.3 and 8.8 p.p.m.) were able to interact with the GFP-Abi2 protein, whereas ANK ⁄ SH3-GST, SAM-GST and PRD3-GST did not show an association It is worth noting that the second proline-rich region showed significantly increased interaction compared with the first, suggesting that PRD2-GST contains the major binding site for Abi2 (Fig 5) This is supported by the finding that Fig Proline-rich regions of Caskin1 interact with Abi2 Lysates of COS7 cells expressing GFP-Abi2 were subjected to affinity purification with the following Caskin1 GST fusion proteins (20 lgỈ point)1) immobilized on glutathione–agarose beads: the ankyrin repeats and the SH3 domain (ANK ⁄ SH3-GST), the SAM domains (SAM-GST) and the three proline-rich regions (PRD1–3-GST) The full-length PRD-GST was also used Bound proteins were eluted by SDS sample buffer, subjected to 7.5% SDS-PAGE, transferred to nitrocellulose and immunoblotted with monoclonal anti-GFP IgG Lysates of COS7 cells immunoblotted with anti-GFP IgG are also shown (bottom panel) Although the exact function of Caskin1 is uncertain, several observations suggest that it probably belongs to the family of scaffold proteins Scaffold proteins are signalling proteins that typically have multiple binding domains for simultaneous interaction with a variety of partners They have no catalytic activity, but tether several signalling proteins to organize them into pathways, thus providing directionality and specificity in signalling For example, the Shank proteins serve as important scaffold molecules modulating signalling pathways at the post-synaptic sites of brain excitatory synapses [36] Ste5 serves in the yeast mating pathway, ensuring that components of the mitogen-activated protein kinase (MAPK) cascade, also involved in osmoresponse and filamentation pathways, act specifically [18] In our case, Caskin1 has been found in a yeast two-hybrid screen to bind about 10 other partners besides Abi2 (Table 1), and several points suggest that it is a bona fide scaffold protein: (a) Caskin1 has a modular structure with several of its domains and non-domain regions involved in protein–protein interactions; (b) none of its domains shows catalytic function; (c) it has 11 different partners all involved in signal transduction; (d) it is preferentially located in the PSD, known to harbour many proteins of signalling and scaffold function (e.g PSD95, Shank, Homer, etc [37]); (e) it has long uncharacterized regions which lack sequence similarity to other proteins, and has been shown here to be intrinsically disordered The appearance and functional role of structural disorder have been explicitly noted in other scaffold proteins, such as Ste5 [18], BRCA1 [19], CBP [4] and Mypt1 [20] Thus, we decided to study this feature in detail to gain further insight into the possible importance of disorder in Caskin1 function and the class of scaffolds in general The collection of scaffold proteins for bioinformatics study, however, is hampered by the lack of consensus on the definition of these proteins In this article, we focus on three classes of complex-forming proteins of related function, also including anchor and docking proteins The prototype for anchor proteins is the FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS 3749 High levels of disorder in scaffold proteins V Csizmok et al Table Results from the two-hybrid screen using a fragment of Caskin1 (amino acids 280–963) as bait and a human fetal cDNA library The numbers in parentheses represent the number of identical clones obtained Clone (12) (2) (1) (1) (2) (1) (1) (1) (1) 10 (7) 11 (12) Protein Function Abl-interactor-2 (Abi2) CASK EphA2 L1CAM Myosin IB Nck1 Neurexin Adaptor protein Scaffold protein Receptor tyrosine kinase Cell adhesion molecule Class I myosin Adaptor protein Neuronal cell adhesion molecule Stathmin family protein Stathmin-like protein Synaptotagmin Septin Siah1 Mediator of Ca2+-regulated vesicle fusion Cell cycle regulator in yeast Ubiquitin ligase A-kinase anchoring protein, which localizes protein kinase A to different subcellular compartments [38] Docking proteins, in general, have an N-terminal membrane targeting element, typically a Pleckstrin homology domain, a myristoylation site or a short transmembrane domain After direct or indirect interactions with a tyrosine kinase, the docking protein becomes tyrosine phosphorylated on multiple sites that can interact with signalling proteins containing SH2 domains Insulin receptor substrate 1, for example, contains an N-terminal Pleckstrin homology domain and a phosphotyrosine-binding domain, and nearly 20 potential tyrosine phosphorylation sites at the C-terminus [39] As suggested above, scaffold proteins are able to interact with many different proteins at the same time, but they are typically not subject to phosphorylation, which creates novel binding sites The lack of consensus on these definitions is also indicated by the sole study addressing the structural disorder in scaffold proteins [2], in which several proteins clearly not of scaffold function (e.g p53, a transcription factor and voltageactivated potassium channel, a binding partner of the scaffold protein PSD95 [40]) were involved Our study encompasses proteins involved in the formation of multiprotein complexes, which have modular organization We collected 74 such proteins by literature search and analysed their disorder by three different algorithms Prediction of disorder in scaffold proteins The structural disorder of the 74 scaffold, docking and anchor proteins was predicted by three different algorithms, i.e IUPred, VSL2 and FoldIndex (Table S1, 3750 see Supporting information) We found that the ratio of residues in local disorder was very high (49.7%, 63.36% and 47.82% predicted by IUPred, VSL2 and FoldIndex, respectively) in these proteins, which is comparable with the ratio found in the most disordered protein families i.e proteins involved in transcription or signal transduction [41] and in RNA chaperones [27] This high level of disorder suggests functional importance in scaffolds Further, we asked whether disorder can be ascribed to regions intervening between the noted functional domains in these proteins To this end, their sequences were analysed to localize their structured PFAM domains Some, described in detail in the literature, are shown in Fig The analysis of regions connecting the domains gave a very high disorder ratio: 61.13%, 77.53% and 54.84% predicted by IUPred, VSL2 and FoldIndex, respectively; in certain proteins, such as GRB2-associated proteins, it exceeded 90% (Table S1) To demonstrate that these intervening regions are not merely there to connect ordered functional domains, we characterized their length distribution in the examined proteins (Fig 7) Although globular domains tend to be short and show a rather normal distribution, with an average length of 86 amino acids, the distribution of linker regions is wide, with an average length of 140 amino acids, and a maximal length as long as 1579 amino acids (in BRCA1) Discussion Our knowledge of the structure of scaffold proteins is largely limited to those regions for which three-dimensional structure has been established However, if we consider that the binding of numerous proteins in tight proximity is rather difficult in the case of a rigid, globular structure, it is reasonable to assume that these proteins contain long, disordered regions Nevertheless, the occurrence of disorder and its functional consequences in scaffold proteins have never been examined systematically The present study provides evidence for the extensive disorder of Caskin1 and also for the class of scaffold proteins in general Overall, the level of disorder exceeds that of the functional class so far considered to be the most disordered: RNA chaperones [27] It is known that, in proteins associated with signal transduction, transcription and RNA chaperone activities, the ratio of amino acids in locally disordered regions is very high, on the order of 50–60% These high levels are thought to result from the functional advantages provided by disorder, which enables functions that cannot be carried out by globular proteins One advantage of the extended, disordered conformation is an FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS V Csizmok et al High levels of disorder in scaffold proteins Fig Schematic representation of the domain structure of selected scaffold proteins The scheme shows the domain architecture of 20 selected scaffold proteins representing 20 families described in detail in the literature established by PFAM Long grey lines connecting the domains are regions with no recognizable similarity to known proteins Fig Length distribution of domains and linker regions in scaffold proteins The numbers of occurrences of domains (light grey) and linker regions (dark grey) with their indicated lengths in the 74 scaffold proteins (Table S1) are given The occurrence was calculated for 50 amino acid length bins, always including the upper limit At the end of the scale, linkers above 800 amino acids in length are grouped (their maximum length extends to 1579 amino acids) enhanced interaction capacity of the protein [12], which is also manifested in the elevated level of disorder of hub proteins [13–16] and the increase in disorder with complex size [17] As disordered regions are often directly involved in protein–protein interactions [42], these points help us to interpret the possible role of disorder in Caskin1 and in other scaffold proteins To obtain a balanced view of structural disorder, it should also be taken into consideration that it may also pose a danger to the cell, such as the occurrence of oncogenic fusion proteins in cancer and amyloid aggregates in neurodegenerative diseases [4] It is probably a result of these adverse effects that the cellular level of IDPs is tightly regulated by several mechanisms [43] Caskin1 is present in the PSD of neuronal cells Within its N-terminal half, it contains some well-characterized domains, which are involved in the interaction with Cask [1], but the C-terminal, proline-rich region has never been examined According to our structural studies, this entire region is intrinsically disordered, and proline-rich regions are known to interact with SH3, WW and other domains of cognate proteins [31,44] Indeed, PRD of Caskin1 contains several consensus SH3 binding sites, and we postulate that it is involved in multiple interactions with other PSD proteins In this study, we have shown that the proline-rich regions interact with the Abi2 protein, which have SH3 domains (we have also found the in vivo association of Abi2 with Caskin1; A Balazs, V Csizmok, P Tompa, R Udupa & L Buday, unpublished results) In this sense, PRD of Caskin1 might function in a manner similar to the long, central, disordered region of BRCA1, which harbours binding motifs for multiple partners in DNA repair [19] A further point on the function of PRD of Caskin1 is that all of our studies point to a local tendency of ordering in the middle PRD2 segment (amino acids 805–1199) The level of hydration of this fragment is lower than that of the other two and the results of CD analysis also show some deviation from a fully disordered, random coil-like state By gel filtration chromatography, this region also shows less extended conformation than the rest of PRD As a local tendency for ordering is a sign of sites poised for interactions [45,46], it FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS 3751 High levels of disorder in scaffold proteins V Csizmok et al is conceivable that this middle segment of PRD in Caskin1 is a primary site of interaction with multiple partners in PSD, especially as PRD2 is the major binding site for Abi2 All of these inferences on the function of Caskin1 are perfectly in line with the organization of PSD PSD is a dynamic multiprotein complex attached to the post-synaptic membrane, composed of several hundred proteins, including receptors and channels, cell adhesion proteins, cytoskeletal proteins, G-proteins and their modulators, and signalling molecules including kinases and phosphatases [47] A variety of scaffold proteins, such as members of the MAGUK, Shank and Homer families, serve to organize PSD As a result of its modular character and ability to form multiprotein interactions, we suggest that Caskin1 is a novel scaffold protein in PSD Previous scattered observations with other scaffold proteins [4,18,19], our novel data on Caskin1 and the noted functional advantages of disorder related to molecular recognition [12,42,48] point towards the general role of disorder in scaffold proteins This inference was underscored by the prediction of disorder for a collection of 74 scaffold proteins: on average, 53.6% of their amino acids were in locally disordered regions Disorder, however, is not evenly distributed in the sequences, as shown by the consideration of only the regions connecting PFAM domains The predicted average disorder for these regions is 64.5%, which suggests that scaffold proteins are constructed as beads on a string from globular domains connected by occasionally very long linker regions Because these linkers cover 65.8% of the total length of scaffold proteins on average, and their average length far exceeds that of the globular domains, there is no doubt that disorder in these proteins fulfils very important functions, probably commensurable in importance with that of ordered domains Actual data on some scaffold proteins provide evidence that these regions are much more than mere passive linkers of functional globular domains For example, BRCA1 contains an approximately 1500amino-acid-long central region between the N-terminal RING domain and C-terminal BRCT domain [19] Although it lacks stable structural elements or recognizable domains, this region is implicated in binding not only DNA, but numerous proteins involved in DNA damage response and repair [49,50] Another scaffold protein, Mypt1, also contains a long disordered segment in its N-terminal region, and this segment is involved in binding to the type protein phosphatase [51] CBP has also been amply characterized in this respect This protein contains seven globular domains and intervening disordered regions At 3752 least two regions of specific partner-binding function, the nuclear receptor interaction domain and the nuclear receptor co-activator-binding domain, reside in the disordered regions of the protein [4] In the case of Ste5, the scaffold protein that binds several kinases of the MAPK pathway, binding of Fus3 has been shown to fall into a locally disordered region [18] These data on scaffold proteins suggest that their long disordered regions present binding sites for their partners As a result of their extended conformation, they have a large potential binding capacity, being able to anchor multiple partners next to each other Interaction sites in disordered regions, termed preformed structural elements [45], linear motifs [48], primary contact sites [52] or molecular recognition features [46], usually only constitute a few residues, and thus enable a very economical and high-capacity binding of partners Furthermore, these regions are often the sites of posttranslational modifications [48,53], and may themselves affect the activity of the bound partner [18], which suggests a rather elaborate and complex binding ⁄ organizing role in the function of scaffold proteins We hope that this suggestion provides novel insight into the function of scaffold proteins, and will instigate the design of novel experimental approaches aimed at resolving the structure and function of these important proteins Materials and methods DNA constructs The full-length rat Caskin1 cDNA was kindly provided by Thomas Sudhof (University of Texas Southwestern Medical ă Center, Dallas, TX, USA), and the full-length Abi2 cDNA was donated by Ann Marie Pendergast (Duke University Medical Center, Durham, NC, USA) Caskin1 cDNA was amplified by a high-fidelity DNA polymerase and subcloned into the pcDNA 3.1 ⁄ V5-His TOPO vector (Invitrogen, San Diego, CA, USA) The full-length Abi2 was amplified by PCR and subcloned into the BamHI site of the pEGFP-C1 vector (BD Biosciences Clontech, San Jose, CA, USA) cDNAs corresponding to the ankyrin repeats and SH3 domain (ANK ⁄ SH3-GST, amino acids 1–346), SAM domains (SAM-GST, amino acids 347–610), proline-rich region (PRD1-GST, amino acids 603–804), proline-rich region (PRD2-GST, amino acids 804–1199), proline-rich region (PRD3-GST, amino acids 1200–1430) and the full-length PRD of Caskin1 (PRD-GST, amino acids 603–1430) were amplified by PCR and subcloned into the EcoRI ⁄ SalI sites of the pGEX-4T1 vector (Amersham Biosciences, Fairfield, CT, USA) as GST fusion proteins FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS V Csizmok et al For pull-down experiments, GST fusion proteins were purified by binding to glutathione–agarose (Sigma, St Louis, MO, USA) without elution Protein purification was monitored on Coomassie blue-stained SDS–PAGE gels: the majority of the GST proteins gave single bands The full-length proline-rich region of Caskin1 (PRD-His) and its fragments (PRD1-His, PRD2-His, PRD3-His) were also subcloned into the BamHI ⁄ XhoI sites of the expression vector pQE2 (Qiagen, Venlo, the Netherlands) with a C-terminal His tag PRD2, because of poor expression of the protein, was further subcloned into the NdeI ⁄ XhoI sites of the expression vector pET20b (Novagen, San Diego, CA, USA) with a C-terminal His tag In all cases, the constructs were verified by DNA sequencing (MWG-Biotech, Ebersburg, Germany) Protein purification For structural characterization, the full-length PRD of Caskin1 and its fragments were expressed in the E coli strain BL21 Star The expression of the proteins was induced by 0.5 mm isopropyl thio-b-d-galactoside at 30 °C for h The proteins were purified to homogeneity from cellular extracts by heat treatment of the supernatants (5 · 100 °C), followed by nickel nitrilotriacetic acid affinity chromatography (Qiagen) For further purification, the dialysed proteins were loaded onto an SP-Sepharose ion exchange chromatograph (Amersham) in a buffer of 20 mm Tris, mm EDTA, pH 7.5, and then eluted by a linear salt gradient (50–500 mm NaCl) Fractions with the highest level of protein were pooled, dialysed into 20 mm Tris, 150 mm NaCl, mm EDTA, pH 7.5 and stored frozen at )20 °C in aliquots The purity of the constructs was demonstrated by SDS-PAGE (Fig S1, see Supporting information) CD measurements CD spectra were recorded at a protein concentration of 0.1 mgỈmL)1 in 10 mm Na2HPO4, 150 mm NaCl, pH 7.5 in a cuvette (path length, mm) on a Jasco J-720 spectropolarimeter (Jasco, Oklahoma City, OK, USA) in a continuous mode with a bandwidth of nm, response time of s and scan speed of 20 nmỈmin)1 All spectra shown were obtained by subtracting the buffer spectrum and averaging 10 separate scans Gel filtration chromatography The unfolded nature of PRD and its fragments was also characterized by gel filtration chromatography The proteins (200 lL) were run on an Amersham Biosciences Superdex 200 (1 · 30 cm) column at 0.5 mLỈmin)1 in a buffer of 50 mm Na2HPO4, 150 mm NaCl, pH 7.0 on an Amersham Biosciences FPLC system The proteins were High levels of disorder in scaffold proteins detected at 280 nm The column was calibrated using the following globular proteins (m in parentheses): ribonuclease A (13.7 kDa), chymotrypsinogen A (25.0 kDa), ovalbumin (43.0 kDa), BSA (67 kDa) and alcohol dehydrogenase (146.8 kDa) The m values of the proteins were determined from the calibration curve constructed by plotting log m values of calibration proteins vs the elution volume The hydrodynamic dimension was characterized by the ratio of mapp, determined by gel filtration chromatography, and the absolute value of m, calculated from the amino acid sequence of the protein NMR spectroscopy H-NMR spectra of PRD1, PRD2 and PRD3 were recorded at 500 MHz on a Bruker DRX instrument (Bruker, Billerica, MA, USA); 16 000 complex data points were acquired in the direct dimension at 300 K using a spectral width of 12 p.p.m Data were zero-filled and processed with a shifted quadratic sinbell plus exponential window function For water suppression, the 3-9-19 pulse sequence with gradients was used [54] Wide-line NMR spectrometry The mobile proton (water) fraction was measured directly by two 1H-NMR methods: by measuring the free induction decay signal or recording Carr–Purcell–Meiboom–Gill echo trains The determination of the mobile water fraction is based on the comparison of the signal intensity or echo amplitude extrapolated to t = with the corresponding values measured at a temperature at which the whole sample is in the liquid state Details of the applied method have been described elsewhere [32,33,55] The effect of freezing on protein solutions was controlled by the comparison of NMR parameters obtained before and after a freeze–thaw cycle at temperatures above °C We found that the freeze–thaw cycle caused no observable changes for the studied samples as far as the measured NMR parameters were concerned The temperature was controlled by an open-cycle Oxford cryostat with a stability of ± 0.1 °C; the uncertainty of the temperature scale was ± °C 1H-NMR measurements and data acquisition were accomplished using a Bruker SXP 4-100 NMR pulse spectrometer at x0 ⁄ 2p = 82.55 MHz with a stability of better than ± 10)6 The data points in the figures are based on spectra recorded by averaging signals to reach a signal to noise ratio of 50 The number of averaged NMR signals was varied to achieve the desired signal quantity for each sample and for unfrozen water quantities The sensitivity of the NMR spectroscope on sample change was controlled by measuring the length of the p ⁄ pulse to obtain reliable M0 values [55] The extrapolation to zero time was performed by fitting a stretched exponential FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS 3753 High levels of disorder in scaffold proteins V Csizmok et al Antibodies and cell lines Acknowledgements Monoclonal antibody raised against GFP was supplied by the Cancer Research UK Hybridoma Development Unit, London, UK COS7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 unitsỈmL)1) and streptomycin (50 lgỈmL)1) This research was supported by grants OTKA K60694 and K61555 from the Hungarian Scientific Research Fund, ETT 245 ⁄ 2006 from the Hungarian Ministry of ´ ´ Health, Mihaly Polanyi Program (Agency for Research Fund Management and Research Exploitation, KPI) and the International Senior Research Fellowship ISRF 067595 from the Wellcome Trust R.U acknowledges support by the Marie Curie RTN ‘ENDOCYTE’ ´ from the European Union FP6 program Peter Banki is acknowledged for technical assistance with wide-line NMR spectrometry Transient transfection Lipofectamine was obtained from Invitrogen and used for the transfection of COS7 cells according to the manufacturer’s instructions Briefly, · 106 cells were plated on to 10 cm Petri dishes 24 h prior to transfection; lg of the various plasmid constructs and 50 lL of lipofectamine were added to each well in mL OptiMEM (Gibco, North Andover, MA, USA) After h, the cells were washed once with DMEM and cultured in their regular medium Protein precipitation and western blotting COS7 cells were washed with ice-cold NaCl ⁄ Pi and lysed in mL of ice-cold 50 mm Hepes buffer, pH 7.4, containing 100 mm NaCl, 1% Triton X-100, 20 mm NaF, mm EGTA, mm Na3VO4, mm p-nitrophenylphosphate, 10 mm benzamidine, mm phenylmethylsulphonyl fluoride and 25 lgỈmL)1 each of leupeptin, soybean trypsin inhibitor and aprotinin The lysates were clarified by centrifugation at 15 000 g for 10 at °C The lysates were then precipitated with 20 lg of the indicated GST-fusion protein immobilized on glutathione–agarose (Sigma) for h at °C Protein precipitates were washed three times with icecold NaCl ⁄ Pi, pH 7.4, containing 0.4% Triton X-100 and eluted with SDS sample buffer Bound proteins were separated by SDS-PAGE and, because of the small amount of proteins, transferred to nitrocellulose membrane and immunoblotted with the indicated antibodies Blots were developed by the enhanced chemiluminescence (ECL; Amersham Biosciences) system Collection of scaffold proteins and bioinformatics predictions We collected a number of anchor, docking and scaffold proteins (denoted collectively as scaffold proteins) from the literature and by screening the UniProt knowledgebase (Table S1) For the prediction of disorder, three different algorithms, i.e IUPred [13,23], VSL2 [24,25] and FoldIndex [26], were used (http://iupred.enzim.hu, http://www.ist.temple.edu/ disprot/predictorVSL2.php, http://bioportal.weizmann.ac.il/ fldbin/findex) The domain prediction was performed by the PFAM algorithm (http://pfam.sanger.ac.uk/) for all the scaffold proteins 3754 References Tabuchi K, Biederer T, Butz S & Sudhof TC (2002) CASK participates in alternative tripartite complexes in which Mint competes for binding with caskin 1, a novel CASK-binding protein J Neurosci 22, 4264– 4273 Cortese MS, Uversky VN & Keith Dunker A (2008) Intrinsic disorder in scaffold proteins: getting more from less Prog Biophys Mol Biol 98, 85–106 Tompa P & Fuxreiter M (2008) Fuzzy complexes: polymorphism and structural disorder in protein–protein interactions Trends Biochem Sci 33, 2–8 Dyson HJ & Wright PE (2005) Intrinsically unstructured proteins and their functions Nat Rev Mol Cell Biol 6, 197–208 Tompa P (2002) Intrinsically unstructured proteins Trends Biochem Sci 27, 527–533 Uversky VN, Oldfield CJ & Dunker AK (2005) Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling J Mol Recognit 18, 343– 384 Sickmeier M, Hamilton JA, LeGall T, Vacic V, Cortese MS, Tantos A, Szabo B, Tompa P, Chen J, Uversky VN et al (2007) DisProt: the Database of Disordered Proteins Nucleic Acids Res 35, D786–D793 Dunker AK, Obradovic Z, Romero P, Garner EC & Brown CJ (2000) Intrinsic protein disorder in complete genomes Genome Inform Ser Workshop Genome Inform 11, 161–171 Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF & Jones DT (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life J Mol Biol 337, 635–645 10 Tompa P, Dosztanyi Z & Simon I (2006) Prevalent structural disorder in E coli and S cerevisiae proteomes J Proteome Res 5, 1996–2000 11 Tompa P (2005) The interplay between structure and function in intrinsically unstructured proteins FEBS Lett 579, 3346–3354 FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS V Csizmok et al 12 Gunasekaran K, Tsai CJ, Kumar S, Zanuy D & Nussinov R (2003) Extended disordered proteins: targeting function with less scaffold Trends Biochem Sci 28, 81–85 13 Dosztanyi Z, Chen J, Dunker AK, Simon I & Tompa P (2006) Disorder and sequence repeats in hub proteins and their implications for network evolution J Proteome Res 5, 2985–2895 14 Ekman D, Light S, Bjorklund AK & Elofsson A (2006) What properties characterize the hub proteins of the protein–protein interaction network of Saccharomyces cerevisiae? Genome Biol 7, R45 15 Haynes C, Oldfield CJ, Ji F, Klitgord N, Cusick ME, Radivojac P, Uversky VN, Vidal M & Iakoucheva LM (2006) Intrinsic disorder is a common feature of hub proteins from four eukaryotic interactomes PLoS Comput Biol 2, e100 16 Patil A & Nakamura H (2006) Disordered domains and high surface charge confer hubs with the ability to interact with multiple proteins in interaction networks FEBS Lett 580, 2041–2045 17 Hegyi H, Schad E & Tompa P (2007) Structural disorder promotes assembly of protein complexes BMC Struct Biol 7, 65 18 Bhattacharyya RP, Remenyi AGood MC, Bashor CJ, Falick AM & Lim WA (2006) The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway Science 311, 822–826 19 Mark WY, Liao JC, Lu Y, Ayed A, Laister R, Szymczyna B, Chakrabartty A & Arrowsmith CH (2005) Characterization of segments from the central region of BRCA1: an intrinsically disordered scaffold for multiple protein–protein and protein–DNA interactions? J Mol Biol 345, 275–287 20 Csizmok V, Szollosi E, Friedrich P & Tompa P (2006) A novel two-dimensional electrophoresis technique for the identification of intrinsically unstructured proteins Mol Cell Proteomics 5, 265–273 21 Namba K (2001) Roles of partly unfolded conformations in macromolecular self-assembly Genes Cells 6, 1–12 22 Dosztanyi Z, Csizmok V, Tompa P & Simon I (2005) IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content Bioinformatics 21, 3433–3434 23 Dosztanyi Z, Csizmok V, Tompa P & Simon I (2005) The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins J Mol Biol 347, 827– 839 24 Peng K, Radivojac P, Vucetic S, Dunker AK & Obradovic Z (2006) Length-dependent prediction of protein intrinsic disorder BMC Bioinformatics 7, 208 High levels of disorder in scaffold proteins 25 Obradovic Z, Peng K, Vucetic S, Radivojac P & Dunker AK (2005) Exploiting heterogeneous sequence properties improves prediction of protein disorder Proteins 61(Suppl 7), 176–182 26 Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O, Beckmann JS, Silman I & Sussman JL (2005) FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded Bioinformatics 21, 3435–3438 27 Tompa P & Csermely P (2004) The role of structural disorder in the function of RNA and protein chaperones FASEB J 18, 1169–1175 28 Li SS (2005) Specificity and versatility of SH3 and other proline-recognition domains: structural basis and implications for cellular signal transduction Biochem J 390, 641–653 29 Mayer BJ (2001) SH3 domains: complexity in moderation J Cell Sci 114, 1253–1263 30 Kim CA & Bowie JU (2003) SAM domains: uniform structure, diversity of function Trends Biochem Sci 28, 625–628 31 Williamson MP (1994) The structure and function of proline-rich regions in proteins Biochem J 297, 249– 260 32 Bokor M, Csizmok V, Kovacs D, Banki P, Friedrich P, Tompa P & Tompa K (2005) NMR relaxation studies on the hydrate layer of intrinsically unstructured proteins Biophys J 88, 2030–2037 33 Tompa P, Banki P, Bokor M, Kamasa P, Kovacs D, Lasanda G & Tompa K (2006) Protein–water and protein–buffer interactions in the aqueous solution of an intrinsically unstructured plant dehydrin: NMR intensity and DSC aspects Biophys J 91, 2243–2249 34 Dyson HJ & Wright PE (2004) Unfolded proteins and protein folding studied by NMR Chem Rev 104, 3607– 3622 35 Dai Z & Pendergast AM (1995) Abi-2, a novel SH3containing protein interacts with the c-Abl tyrosine kinase and modulates c-Abl transforming activity Genes Dev 9, 2569–2582 36 Sheng M & Kim E (2000) The Shank family of scaffold proteins J Cell Sci 113 (Pt 11), 1851–1856 37 Feng W & Zhang M (2009) Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density Nat Rev Neurosci 10, 87–99 38 Pawson T & Scott JD (1997) Signaling through scaffold, anchoring, and adaptor proteins Science 278, 2075–2080 39 Thirone AC, Huang C & Klip A (2006) Tissue-specific roles of IRS proteins in insulin signaling and glucose transport Trends Endocrinol Metab 17, 72–78 40 Magidovich E, Orr I, Fass D, Abdu U & Yifrach O (2007) Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS 3755 High levels of disorder in scaffold proteins 41 42 43 44 45 46 47 48 49 50 V Csizmok et al interaction with scaffold proteins Proc Natl Acad Sci USA 104, 13022–13027 Iakoucheva LM, Brown CJ, Lawson JD, Obradovic Z & Dunker AK (2002) Intrinsic disorder in cell-signaling and cancer-associated proteins J Mol Biol 323, 573–584 Dyson HJ & Wright PE (2002) Coupling of folding and binding for unstructured proteins Curr Opin Struct Biol 12, 54–60 Gsponer J, Futschik ME, Teichmann SA & Babu MM (2008) Tight regulation of unstructured proteins: from transcript synthesis to protein degradation Science 322, 1365–1368 Kay BK, Williamson MP & Sudol M (2000) The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains FASEB J 14, 231–241 Fuxreiter M, Simon I, Friedrich P & Tompa P (2004) Preformed structural elements feature in partner recognition by intrinsically unstructured proteins J Mol Biol 338, 1015–1026 Vacic V, Oldfield CJ, Mohan A, Radivojac P, Cortese MS, Uversky VN & Dunker AK (2007) Characterization of molecular recognition features, MoRFs, and their binding partners J Proteome Res 6, 2351–2366 Beresewicz M (2007) Scaffold proteins (MAGUK, Shank and Homer) in postsynaptic density in the central nervous system Postepy Biochem 53, 188–197 Fuxreiter M, Tompa P & Simon I (2007) Structural disorder imparts plasticity on linear motifs Bioinformatics 23, 950–956 Wang Q, Zhang H, Kajino K & Greene MI (1998) BRCA1 binds c-Myc and inhibits its transcriptional and transforming activity in cells Oncogene 17, 1939–1948 Zhang H, Somasundaram K, Peng Y, Tian H, Bi D, Weber BL & El-Deiry WS (1998) BRCA1 physically associates with p53 and stimulates its transcriptional activity Oncogene 16, 1713–1721 3756 51 Toth A, Kiss E, Herberg FW, Gergely P, Hartshorne DJ & Erdodi F (2000) Study of the subunit interactions in myosin phosphatase by surface plasmon resonance Eur J Biochem 267, 1687–1697 ´ 52 Csizmok V, Bokor M, Banki P, Klement E, Medzihradszky KF, Friedrich P, Tompa K & Tompa P (2005) Primary contact sites in intrinsically unstructured proteins: the case of calpastatin and microtubule-associated protein Biochemistry 44, 3955–3964 53 Iakoucheva LM, Radivojac P, Brown CJ, O’Connor TR, Sikes JG, Obradovic Z & Dunker AK (2004) The importance of intrinsic disorder for protein phosphorylation Nucleic Acids Res 32, 1037–1049 54 Piotto M, Saudek V & Sklenar V (1992) Gradienttailored excitation for single-quantum NMR spectroscopy of aqueous solutions J Biomol NMR 2, 661–665 55 Tompa K, Banki P, Bokor M, Lasanda G & Vasaros L (2003) Diffusible and residual hydrogen in amorphous Ni(Cu)-Zr-H alloys, J Alloys Comp 350, 52–55 Supporting information The following supplementary material is available: Fig S1 SDS-PAGE with the various forms of Caskin1 with the different tags Table S1 Predicted disorder of scaffold proteins This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS ... C-terminal Caskin1 are functional and may interact with SH3 domain-containing proteins, such as Abi2 [We have also found an in vivo association and colocalization of Abi2 with Caskin1 (A Balazs,... of Caskin1 fragments As described in the introductory paragraphs, the N-terminal half of Caskin1 contains a number of well-known domains involved in protein–protein interaction, such as the ankyrin... folded structural domains, we anticipated that structural disorder may be a general feature of scaffold proteins In a recent review, structural disorder in several scaffold proteins and in other proteins