Protein Disorder Prediction

Rune Linding; Lars Juhl Jensen; Francesca Diella; Peer Bork; Toby J Gibson; Robert B Russell

doi:10.1016/J.STR.2003.10.002

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Protein Disorder Prediction

Francesca Diella

2003, Structure

https://doi.org/10.1016/J.STR.2003.10.002

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7 pages

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Abstract

It is becoming increasingly clear that many functionally important protein segments occur outside of globu-Biocomputing Unit Meyerhofstr 1 lar domains (Wright and Dyson, 1999; Dunker et al., 2002). Protein structure and function space is parti-D-69117 Heidelberg Germany tioned in two subspaces. The first consist of globular units with binding pockets, active sites, and interaction 2 Max-Delbrü ck-Centre fü r Molecular Medicine Robert-Rö ssle-Strasse 10 surfaces. The second subspace contains nonglobular segments such as sorting signals, posttranslational modi-D-13092 Berlin Germany fication sites, and protein ligands (e.g., SH3 ligands). Globular units are built of regular secondary structure 3 CellZome GmbH Meyerhofstr 1 elements and contribute the majority of the structural data deposited in PDB. In contrast, the nonglobular sub-D-69117 Heidelberg Germany space encompasses disordered, unstructured and flexible regions without regular secondary structure. Functional sites within the nonglobular space are known as linear motifs (cataloged by ELM [http://elm.eu.org]) Summary (Puntervoll et al., 2003). There are also many recent reports of Intrinsically A great challenge in the proteomics and structural genomics era is to predict protein structure and func-Disordered Proteins (IDPs, also known as Intrinsically Unstructured Proteins). These are proteins or domains tion, including identification of those proteins that are partially or wholly unstructured. Disordered regions in that, in their native state, are either completely disordered or contain large disordered regions. More than proteins often contain short linear peptide motifs (e.g., SH3 ligands and targeting signals) that are important 100 such proteins are known including Tau, Prions, Bcl-2, p53, 4E-BP1, and eIF1A (see Figure 4) (Tompa, for protein function. We present here DisEMBL, a computational tool for prediction of disordered/unstruc-2002; Uversky, 2002). Protein disorder is important for understanding pro-tured regions within a protein sequence. As no clear definition of disorder exists, we have developed pa-tein function as well as protein folding pathways (Plaxco and Gross, 2001; Verkhivker et al., 2003). Although little rameters based on several alternative definitions and introduced a new one based on the concept of "hot is understood about the cellular and structural meaning of IDPs, they are thought to become ordered only when loops," i.e., coils with high temperature factors. Avoiding potentially disordered segments in protein expression bound to another molecule (e.g., CREB-CBP complex [Radhakrishnan et al., 1997]) or owing to changes in constructs can increase expression, foldability, and stability of the expressed protein. DisEMBL is thus the biochemical environment (Dunker et al., 2001, 2002; Uversky, 2002). useful for target selection and the design of constructs as needed for many biochemical studies, particularly The current view on disorder is that disordered proteins are disordered to allow for more interaction part-structural biology and structural genomics projects. The tool is freely available via a web interface (http:// ners and modification sites (Wright and Dyson, 1999; Liu et al., 2002; Tompa, 2002). It has also been suggested dis.embl.de) and can be downloaded for use in largescale studies. that disordered proteins exist to provide a simple solution to having large intermolecular interfaces while keeping smaller protein, genome and cell sizes (Gunasekaran

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The reversible unfolding of metallo-␤-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn 2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-␤-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn 2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger ␣-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn 1-and Zn 2-BcII was carried out. The region spanning the metal binding metallo-␤-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N-and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199m Hg-perturbed angular correlation of ␥-rays spectroscopic data. We suggest that this flexible "zinc arm" motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an "activation on demand" regulation of enzyme activity. The production of metallo-␤-lactamases (MBLs) 2 is one of the defense strategies of bacteria against ␤-lactam antibiotics. MBLs hydrolyze the C-N bond of the ␤-lactam ring of these compounds using protein-bound zinc ions as cofactors (1). Their emergence in pathogenic bacterial strains and their broad substrate profile make them clinically important (2). Whereas the overall structure of all known MBLs is very similar (3), distinct differences in the set of protein ligands for bound zinc ions led to the classification into subclasses B1-B3 (4). Here we have studied the two subclass B1 enzymes BcII and BlaB from Bacillus cereus strain 569/H/9 and Chryseobacterium meningosepticum, respectively, which show 35.2% identical residues (5). The very similar structure of these enzymes is organized in a ␣␤␤␣ sandwich (6, 7). The N-and C-terminal domains are connected by an external loop, and the active site is located in a long channel between the two domains. The binuclear zinc binding site is composed of a 3-His (3H) site and a Asp-Cys-His (DCH) site. Three metal ion ligands are located on the N-terminal domain and constitute the HXHXD motif, which is strictly conserved in proteins of the MBL super family (8). The three remaining metal ligands are located on the C-terminal domain of the proteins. Both for the native and for the cadmium-substituted enzyme, it has been shown that a single metal ion, when bound to BcII, appears to be distributed between the metal binding sites (9-11). The metal ion requirement for catalytic activity of the three subclasses B1-B3 of MBLs is heavily debated. Although most crystal structures of subclass B1 enzymes show binuclear zinc sites (3), it was found that BcII from B. cereus 569/H/9, CcrA from Bacteroides fragilis, BlaB from C. meningosepticum, IMP-1 from Pseudomonas aeruginosa, and L1 from Stenotrophomonas maltophilia are both active as the mono-and di-zinc enzymes (9, 12-15). Recently a study with Co(II)-substituted BcII challenged this view in concluding that only the di-Co-enzyme might be catalytically active (16). The same authors came to the conclusion that also native BcII requires two bound zinc ions for activity (17). A very recent study on the Co(II)-substituted enzyme came to the conclusion that both the Co 1-and the Co 2-enzymes are catalytically active with the DCH site as the primary catalytic site (18).

NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo-and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo-or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks. The regulatory machinery that controls progression through the cell cycle is highly conserved in eukaryotic evolution. In the fungi Aspergillus nidulans the Ser/Thr protein kinase NIMA (Never in Mitosis Gene A) plays a pivotal role in controlling entry into mitosis 1-3. Eleven NIMA-related protein kinases (NEKs) are expressed in humans. While the majority of the different mammalian NEKs still have their roles not fully elucidated, NEK2, NEK6, NEK7 and NEK9 have a well-established role in the regulation of mitosis, especially in centrosome disjunction and mitotic spindle assembly and function 4. NEK1 and NEK8 have also been shown to be involved in the regulation of cilia and NEK1, NEK4, NEK8, NEK10, and NEK11 modulate the DNA damage response 5, 6. In general, mitotic protein kinases such as NEKs have been implicated in guarding the integrity of the genome. NEK1 contains an N-terminal kinase domain and an extended C-terminal domain, with several predicted coiled-coil (CC) regions 7 in which many of the interactions with other proteins occur 6, 8. NEK1 has important regulatory functions during embryogenesis and mice lacking NEK1 have a form of polycystic kidney disease (PKD) 9. These mice lacking NEK1 show pleiotropic malfunctions, including facial dysmorphism, male sterility, dwarfism and anemia. NEK1 regulates cilium assembly 10 and may further link cilia functions to cell-cycle regulation 11. There is an evolutionary relationship between organisms possessing NEK genes and regulation of cilia 12. Furthermore, two mutations in the kinase domain of NEK1 (G145R and L253S) have been associated with short-rib thoracic dysplasia, an autosomal recessive ciliopathy 13. Recently, NEK1 protein variants have been linked to further genetic disorders such as Mohr-syndrome 14 and amyotrophic lateral sclerosis 15. At the protein level, it has been shown that NEK1 stabilizes the complex between ATR (ATM and Rad3-related) and ATRIP (ATR interacting protein) through phosphorylation, priming this complex for Chk1

Multi-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca 2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca 2+ and Na + uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical 'pore-only' proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels. Voltage-gated ion channels selective for Ca 2+ (Ca V), Na + (Na V) and K + (K V) perform a plethora of functions in both excitable and non-excitable cells. Mutations in these channels are the causal basis of numerous diseases, thereby rendering them clinically-relevant drug targets 1. They are composed of four domains that form a central pore, with peripheral voltage sensors. Each domain consists of six transmembrane helices comprising the voltage sensor (S1-S4) and pore (S5-S6) regions. In K V and prokaryotic Na V , the domains are separate subunits that form a tetramer, resulting in symmetrical pores. In contrast, eukaryotic Ca V and Na V are single polypeptide chains with four divergent domains, giving rise to asymmetric pores 1,2. This architectural similarity suggests an evolutionary trajectory whereby a primordial gene encoding a one-domain channel underwent two rounds of intragenic duplication and divergence to generate the extant four-domain channels (Fig. 1A) 3,4. Two-pore channels (TPCs) are less well characterised members of the voltage-gated ion channel superfamily that, unusually, localise to intracellular acidic Ca 2+ stores 5. In animals, they are activated by the second messenger NAADP to release Ca 2+ from the endo-lysosomal system, and are an important part of the cellular signalling apparatus 6-8. Furthermore, TPCs are rapidly emerging as potential therapeutic targets 9-11. Recent crystal structures of a plant TPC 12-14 have confirmed earlier biochemical reports that they form dimers from two-domain (DI and DII) subunits 15,16. This structural organisation identifies TPCs as a key intermediate in the evolution of voltage-gated ion channels from one-domain to four-domain channels (Fig. 1A). Indeed, phylogenetic analyses of the individual TPC domains supports this conclusion, indicating that they are substantially diverged from one another, and are instead more related to equivalent domains in four-domain channels 17. The modularity of the pore regions in symmetrical (often prokaryotic) channels is established 18-21. For example, the isolated pore of a Na V from a marine bacterium forms an open, folded tetramer that is constitutively active, thereby supporting Na + flux in the absence of the voltage sensor 22. Similar results have been found for 'pore-only' proteins derived from other prokaryotic channels 18,20,23. The functional architecture of asymmetric ion channel

Background: PcrV is a hydrophilic translocator of type three secretion system (TTSS) and a structural component of the functional translocon. C-terminal helix of PcrV is essential for its oligomerization at the needle tip. Conformational changes within PcrV regulate the effector translocation. PcrG is a cytoplasmic regulator of TTSS and forms a high affinity complex with PcrV. C-terminal residues of PcrG control the effector secretion. Result: Both PcrV and PcrG-PcrV complex exhibit elongated conformation like their close homologs LcrV and LcrG-LcrV complex. The homology model of PcrV depicts a dumbbell shaped structure with N and C-terminal globular domains. The grip of the dumbbell is formed by two long helices (helix-7 and 12), which show high level of conservation both structurally and evolutionary. PcrG specifically protects a region of PcrV extending from helix-12 to helix-7, and encompassing the C-terminal globular domain. This fragment ΔPcrV (128-294) interacts with PcrG with high affinity, comparable to the wild type interaction. Deletion of N-terminal globular domain leads to the oligomerization of PcrV, but PcrG restores the monomeric state of PcrV by forming a heterodimeric complex. The N-terminal globular domain (ΔPcrV (1-127)) does not interact with PcrG but maintains its monomeric state. Interaction affinities of various domains of PcrV with PcrG illustrates that helix-12 is the key mediator of PcrG-PcrV interaction, supported by helix-7. Bioinformatic analysis and study with our deletion mutant ΔPcrG (13-72) revealed that the first predicted intramolecular coiled-coil domain of PcrG contains the PcrV interaction site. However, 12 N-terminal amino acids of PcrG play an indirect role in PcrG-PcrV interaction, as their deletion causes 40-fold reduction in binding affinity and changes the kinetic parameters of interaction. ΔPcrG (13-72) fits within the groove formed between the two globular domains of PcrV, through hydrophobic interaction. Conclusion: PcrG interacts with PcrV through its intramolecular coiled-coil region and masks the domains responsible for oligomerization of PcrV at the needle tip. Also, PcrG could restore the monomeric state of oligomeric PcrV. Therefore, PcrG prevents the premature oligomerization of PcrV and maintains its functional state within the bacterial cytoplasm, which is a prerequisite for formation of the functional translocon.

The regulation of protein function is often achieved through post-translational modifications including phosphorylation, methylation, ubiquitination, and acetylation. The role of acetylation has been most extensively studied in the context of histones, but it is becoming increasingly evident that this modification now includes other proteins. The Sir2 family of NAD-dependent deacetylases was initially recognized as mediating gene silencing through histone deacetylation, but several family members display non-nuclear subcellular localization and deacetylate non-histone protein substrates. Although many structural and enzymatic studies of Sir2 proteins have been reported, how substrate recognition is achieved by this family of enzymes is unknown. Here we use in vitro deacetylase assays and a variety of potential substrates to examine the substrate specificity of yeast homologue Hst2. We show that Hst2 is specific for acetyl-lysine within proteins; it does not deacetylate small polycations such as acetyl-spermine or acetylated amino termini of proteins. Furthermore we have found that Hst2 displays conformational rather than sequence specificity, preferentially deacetylating acetyl-lysine within unstructured regions of proteins. Our results suggest that this conformational requirement may be a general feature for substrate recognition in the Sir2 family. Protein phosphorylation has long been accepted as a key mechanism in the regulation of diverse cellular processes. Lately, other post-translational modifications including methylation, ubiquitination, and particularly, acetylation, are being recognized as playing key roles in protein function (1). Histone acetylation, for example, mediated by the interplay between histone acetyltransferases and histone deacetylases (HDACs), 2 has proven to be vital for control of gene silencing, transcription, replication, and repair (2). The Sir2 family of NAD-dependent HDACs has homologues in organisms ranging from bacteria to human (3). Since histones are absent in bacteria, it seems likely that the activity of this family is not restricted to histones, for example some family members display cellular localization outside of the nucleus and deacetylate nonhistone protein substrates (3). It is as yet unclear how this family of enzymes achieves its broad substrate specificity. Here we demonstrate that the yeast homologue Hst2 displays conformational rather than sequence specificity, deacetylating acetyl-lysine within unstructured regions of proteins. This suggests that conformational specificity may be a substrate determinant for all members of the Sir2 family. MATERIALS AND METHODS Expression and Purification of Recombinant Proteins-Yeast HST2 and HOS3 open reading frames were amplified by PCR and cloned into pET30a at the XhoI/KpnI and BamHI/HinDIII restriction sites, respectively, to produce NH 2-terminal His 6-tag fusion proteins. Plasmids were transformed into BL21(DE3) cells, and the cells were grown at 37°C to log phase. HST2 expression was induced by the addition of 0.1 mM isopropyl ␤-D-thiogalactopyranoside and overnight incubation at 18°C. HOS3 expression was induced by addition of 1 mM isopropyl ␤-D-thiogalactopyranoside and overnight incubation at 30°C. Cells were harvested and lysed by sonication and protein purified by nickel-chelate chromatography. Hst2 lysate was bound in 50 mM HEPES-KOH, pH 8.0, 300 mM NaCl, 10 M ZnCl 2 , 10 mM imidazole, 1 mM 2-mercaptoethanol, and protease inhibitor-EDTA mixture (Roche Applied Science); eluted in 50 mM HEPES-KOH, pH 8.0, 300 mM NaCl, 10 M ZnCl 2 , 1 mM 2-mercaptoethanol, 300 mM imidazole; and dialyzed into 10 mM HEPES-KOH pH 8.0, 10 M ZnCl 2 , 1 mM dithiothreitol, 10% glycerol. Hos3 was bound in 500 mM NaCl, 20 mM Tris, pH 8.0, 1 mM phenylmethylsulfonyl fluoride; eluted in 500 mM NaCl, 20 mM Tris pH 8.0, 10 M ZnCl 2 , 300 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride; and dialyzed into 500 mM NaCl, 20 mM Tris, pH 8.0, 10 M ZnCl 2 , 10 mM 2-mercaptoethanol. The purity of both proteins was estimated to be Ͼ90% by SDS-PAGE and Coomassie Blue staining. Expression and Purification of TAP-tagged Proteins-TAP containing yeast strains were a gift from J. Greenblatt. All strains were grown in YPD (1% yeast, 2% peptone, 2% glucose) to log phase, harvested, and then lysed by vortexing with glass beads and the TAP-tagged protein purified as described (4). Beads were resuspended in deacetylase assay buffer described below. Assays were carried out directly on TAP protein bound to IgG-Sepharose for 3 h at 30°C. Preparation of Acetylated Substrates-Peptides corresponding to the NH 2-terminal sequences of the yeast core histones (SCH2B1, SAKAE-KKPASKAPAEKKPAAC; SCH2A12, SGGKGGKAGSAAKASQSR-SAC; SCH3, ARTKQTARKSTGGKAPRKQLASKAC; SCH4, SGRGK-GGKGLGKGGAKRHRC) were synthesized by a PerSeptive Biosystems Pioneer peptide synthesizer. Crude peptide was purified by reverse phase high performance liquid chromatography on a C18 column using a linear gradient of water-acetonitrile containing 0.06% trifluoroacetic acid. Melittin (M7129), protamine (P3880), cytochrome c (C7752), poly-L-lysine (P0879), poly-D-lysine (P0296), and spermine were purchased from Sigma. Urotensin II was purchased from American Peptide Co. Substrates were lightly acetylated by means of sulfo-N-hydroxysuccinimidyl [ 14 C]acetate or [ 13 C]acetate, which specifically reacts with lysine, and purified as described previously (5). 14 C-Acetylated substrate concentration was determined by scintillation counting. Peptide concentration was determined by microbiuret protein assay (6). Preparation of Denatured Proteins-RNase A was reduced and denatured by incubation for 30 min at 37°C in 8 M urea, 37.5 mM Tris, pH 8.8, 10 mM DTT and then alkylated by addition of 5 mM iodoacetamide and

Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules. Migration through extracellular matrices requires protrusion at the cell front that is mediated by integrin adhesions 1 . The ERC/ELKS scaffold proteins and their partners liprin-α and LL5 2,3 are regulators of a number of important cellular processes including cell migration and invasion 4,5 , the assembly of presynaptic active zones 6 and cortical platforms linked to microtubules . In migrating cells ERC, liprin-α and LL5 proteins form polarized plasma membrane-associated platforms (PMAPs) 9 near the cell edge or near invadosomes 5,10 , to promote the turnover of adhesions/invadosomes and stimulate protrusion 11 . These scaffolds are distinct from exocytic/endocytic markers 5 . Supramolecular markers may harness liquid-liquid phase separation, giving rise to membrane-less organelles with specific functions within nucleus and cytoplasm 12 . Phase-separated systems may help the cell organizing molecules and reactions in space and time . Interestingly, the propensity to undergo phase transition may be favored by intrinsically disordered protein regions (IDRs) characterized by lack of stable structure and increased flexibility . The dynamic accumulation of ERC1/ELKS at sites of surface cell remodeling, and the lack of colocalization with membrane markers, led us to hypothesize that ERC1/ELKS may serve as a scaffold to assemble cytoplasmic condensates that include other components of a protein network relevant to cell motility and to other important cellular functions. Here we show that ERC1 can drive the formation of membrane-less condensates with liquid properties, and that ERC1-mediated condensates specifically host partners of a network relevant to cell motility, including liprin-α1. In this study we have used three types of cells: MDA-MB-231 human breast cancer cells and HT1080 human fibrosarcoma cells were used as examples of migratory cells accumulating ERC1 at the protruding leading edge, where ERC1 and associated proteins are known to play an important role in the regulation of cell edge dynamics ; while COS7 cells were used as a simple experimental system to characterize the formation and behavior of ERC1-positive condensates.

Protein Disorder Prediction

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