Nucleotide-binding mechanisms in pseudokinases

Biosci. Rep. (2016) / 36 / art:e00282 / doi 10.1042/BSR20150226 Nucleotide-binding mechanisms in pseudokinases Henrik M. Hammar´ en*1, Anniina T. Virtanen* and Olli Silvennoinen*†1 *School of Medicine, University of Tampere, Biokatu 8, FI-33014 Tampere, Finland †Clinical Hematology, Department of Internal Medicine, Tampere University Hospital, Medisiinarinkatu 3, FI-33520 Tampere, Finland Synopsis Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10 % of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades. Furthermore, several pseudokinases have been linked to human diseases, particularly cancer, which is raising interest for therapeutic approaches towards these proteins. The ATP-binding pocket is a well-established drug target and elucidation of the mechanism and properties of nt binding in pseudokinases is of significant interest and importance. Recent studies have demonstrated that members of the pseudokinase family are very diverse in structure as well as in their ability and mechanism to bind nts or perform phosphoryl transfer reactions. This diversity also precludes prediction of pseudokinase function, or the importance of nt binding for said function, based on primary sequence alone. Currently available data indicate that ∼40% of pseudokinases are able to bind nts, whereas only few are able to catalyse occasional phosphoryl transfer. Pseudokinases employ diverse mechanisms to bind nts, which usually occurs at low, but physiological, affinity. ATP binding serves often a structural role but in most cases the functional roles are not precisely known. In the present review, we discuss the various mechanisms that pseudokinases employ for nt binding and how this often low-affinity binding can be accurately analysed. Key words: ATP, kinase activity, kinome, nucleotide binding, pseudokinase, signalling. Cite this article as: Bioscience Reports (2016) 36, e00282, doi:10.1042/BSR20150226 INTRODUCTION In their landmark paper in 2002, Manning and colleagues presented for the first time a comprehensive catalogue of human protein kinases (PKs) [1]. One of the salient findings of their analysis was that ∼10% of the 518 human PKs lack at least one of the classical conserved catalytic residues described by Hanks et al. [2]: the β3 lysine (K in the ‘VAIK’ consensus motif, Figure 1), the catalytic aspartate (D in ‘HRD’) or the cation-binding aspartate (D in ‘DFG’). Some of these kinases, like the WNK (‘With no lysine’) family, were already known to have phosphoryl transfer activity despite the lack of canonical catalytic ............................................................................................................................................................................................................................................................................................................ Abbreviations: ADCK, aarF domain-containing protein kinase; AMP-PNP, adenylate-imidodiphosphate; ANK, ankyrin repeat domain (in RNase L); ANPa/ANPb, atrial natriuretic peptide receptor type A/B, also known as atrial natriuretic peptide receptor 1 (NPR1) and NPR2, respectively. Also known as GC-A/GC-B; ATPγS, adenosine 5-[γ-thio]triphosphate; BAK1, BCL2-antagonist/Killer 1; BIR2, BAK1-interacting receptor-like kinase 2; BSK8, brassinosteroid signalling kinase 8; CaM-kinase, Ca2+/calmodulin-dependent protein kinase; CASK, calcium/calmodulin-dependent serine protein kinase; COP1, constitutive photomorphogenesis protein 1; EGFR, epidermal growth factor receptor; ePK, eukaryotic protein kinase; GC, guanylate cyclase; GUCY2, guanylate cyclase, e.g. GUCY2C, also known as GC-C, and heat stable enterotoxin receptor (HSER); HER3, human epidermal growth factor 3, also known as ERBB3; IRAK, interleukin-1 receptor-associated kinase; ITC, isothermal titration calorimetry; JAK, Janus kinase; JH, JAK homology; LKB1, liver kinase B1; MLKL, mixed lineage kinase domain-like; nt, nucleotide; NTE, N-terminal extension; PAN3, PAB1P-dependent poly(A)-nuclease; PK, protein kinase; PDZ, protein domain found in PSD95, Dlg1 and zo-1; PKA, cAMP-dependent protein kinase A; PKD, pseudokinase domain; PKL, protein kinase-like; RIP3, receptor-interacting serine-threonine kinase 3; RLK, receptor-like kinase; ROPK, rhoptry kinase; ROR, receptor tyrosine kinase-like orphan receptor; RTK, receptor tyrosine kinase; SH3, Src-homology 3 domain; SPR, surface plasmon resonance; STRAD, Ste20-related adaptor; TK, titin kinase; TLR, Toll-like receptor; TSA, thermal shift assay; VRK, vaccinia-related kinase; WNK, With no lysine. 1Correspondence may be addressed to either of these authors (email [email protected] or [email protected]). sites. Yet, the other 50 identified proteins or protein domains were deemed likely to be inactive and consequently dubbed ‘pseudokinases’. Subsequently, also some of these pseudokinases (like Haspin) were shown to be catalytically active, and thus could be reclassified as atypical PKs [3]. For these and some more recent examples of catalytic activity like KSR2 [4], HER3 [5], JAK2 JH2 [6]orCASK[7] the distinction of pseudokinase and atypical kinase has become somewhat unclear. However, as proposed by Eyers and Murphy [8], the bioinformatic definition of a pseudokinase (with the inclusion of kinases that have experimentally been found to be inactive) should be maintained for the sake of clarity, and will be used in the present review as well. c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 1 H.M. Hammar´ en, A.T. Virtanen and O. Silvennoinen Figure 1 Conserved motifs and residues contributing to nt binding and kinase activity in (pseudo)kinases (A) Schematic depiction of the secondary structure elements of ePKs. Relative sizes and positions of elements are based on PKA (PDB: 4WB5). (B) Sequence alignment of selected pseudokinases and PKL proteins classified based on current information on nt binding. Conserved PK regions relevant to nt binding are shown. The six highly conserved residues contributing directly to nt binding or catalytic activity are highlighted. *The gatekeeper residue is not part of the 10 conserved kinase residues identified in [14]. All sequences represent human proteins unless otherwise noted. The sequence alignment was made using Clustal W [137,138] and manually corrected based on crystal structures and previous alignments [32,92,139] where available. For non-ePKs the sequences shown are the (predicted) functional/structural equivalents of the conserved residue in question (secondary structure of e.g. ADCK3 or Fam20 kinases is different from the one shown in A). Fam20C is included as an example of an active Fam20 kinase. (C) 3D structure of human PKA (PDB: 4WB5) shown as an example of an archetypal ePK. ATP is shown in sticks and the two magnesium cations as purple spheres. Colours of secondary structure elements are as in (A). Pseudokinases are spread throughout all PK families [1], indicating that at least most of them have emerged repeatedly from active kinases throughout evolution. Despite the catalytically inactive nature of most pseudokinases studied thus far, a recent survey found that 13 out of the 31 diverse pseudokinases studied are still able to bind nts [9]. Further studies on the characteristics and functions of nt binding in pseudokinases have shed light on this unexpected finding, and shown that pseudokinases employ diverse mechanisms to bind ATP and suggest that nt binding can play an important structural and functional role. Several excellent reviews on the advances in the pseudokinase field have been written [3,8,10], including the potential importance of pseudokinases in human disease [11–13]. Recent mounting evidence of the importance of the nt-binding site of pseudokinases in particular, however, warrants a focused discussion on this aspect. The present review aims to provide such a comprehensive update on the heterogeneity of nt binding in pseudokinases. MOTIFS INVOLVED IN NT BINDING IN PK DOMAINS Pseudokinases belong to the protein kinase-like (PKL) superfamily which share a conserved fold with an N lobe comprised mostly of βstrands and a mostly α-helical C lobe (Figure 1C). The catalytic mechanism in PKL proteins involves 10 residues [14] that are highly conserved even if the proteins may otherwise .......................................................................................................................................................................................................................................................................................................................................................................... 2c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. Nucleotide binding in pseudokinases lack sequence conservation [14,15]. Six out of these 10 residues are directly involved in nt or substrate binding or catalysis [14] (highlighted in Figure 1B), whereas the function of the remaining four residues is still not completely understood [14], but is probably mainly structural [16]. The first of the critical residues for nt binding is Gly52 (as the de facto archetypal protein kinase, human cAMP-dependent protein kinase A (PKA) is usually used for numbering of amino acid residues in protein kinase motifs and will also be used in the present review) in the glycine-rich loop (‘Gly-rich loop’, also known as the ‘phosphate-binding loop’ or ‘P-loop’) located between strands β1andβ2(Figure 1). The function of the Glyrich loop is best understood in PKA, in which glycine at this position enables the peptide backbone of the tip of the loop (Ser53)tobindtheγ-phosphate of ATP. Mutation of the Glyrich loop, and especially Gly52, lowers affinity towards ATP and affects kinase activity [17]astheγ-phosphate can no longer be efficiently positioned for phosphoryl transfer between the tip of the Gly-rich loop and a basic residue (Lys168) from the so-called ‘catalytic loop’ situated between β6andβ7(Figure 1)[18]. Although the function of the Gly-rich loop is well known for PKA, the motif has not been extensively studied in other kinases and it is unclear how universal this function is. The second residue is Lys72 of the ‘VAIK’ motif in β3, which is the most conserved residue in all PKL proteins, and the only residue not missing in any of the known families (Figure 1)[14]. Despite its virtually universal conservation and its position next to the αand βphosphates of ATP (Figure 2, PKA), the function of Lys72 in nt binding is not entirely clear, and it has been reported to be dispensable for nt binding in multiple canonical kinases [19– 22]. However, the lysine seems to be required for nt binding in the pseudokinases GUCY2C [23], HER3 [5], TRIB2 [24] and murine (and to a lesser extent also human) MLKL [25,26]. Lys72 (or another lysine in its spatial position, like Lys223, WNK1 in the WNK family [27]) is absolutely required for catalytic activity both in multiple canonical kinases [19,20,22], as well as in the lowactivity pseudokinase JAK2 JH2 [6]. Even though its function in ATP binding is somewhat unclear, Lys72 is critical in making a salt bridge to the conserved Glu91 in the C helix (αC) (Figure 2, PKA), thus linking αC to the nt-binding pocket and the helix in the ‘in’ position, which is a hallmark of the active kinase conformation [16,28]. The last three of the six conserved residues are required for catalysis: Asp166 (‘HRD’) and Asn171 in the catalytic loop, and Asp184 in the ‘DFG’ motif (Figure 1). Asn171 and Asp184 participate in the binding of the two divalent cations accompanying ATP in the canonical mode of ATP binding in kinases (Figure 2, PKA). Similarly to the β3 lysine, Asp166 and Asp184 are not absolutely required for ATP binding, but rather needed for efficient catalysis [22], Asp166 being the catalytic base in the phosphoryl transfer reaction [29]. In addition to these conserved single residues, the purine pocket of the nt-binding site is lined with a group of hydrophobic residues from β2(Val 57)andβ3(Ala 70) from the N lobe, and β7 (Leu173) from the C lobe (Figure 2, PKA). These residues are part of the so-called ‘catalytic spine’ (C spine), which is a conserved hydrophobic structure typically found in active kinases [16,30,31], and which is completed upon binding of a nt’s purine ring between the N and C lobes (shown in light blue in Figure 2). Additionally, PKs also have another nonlinear, conserved structural element called the ‘regulatory spine’ (R spine) [16,30,31], made up of four residues from DFG, HRD, αC and the αC-β4 loop (shown in beige in Figure 2). This structure is stabilized by phosphorylation of the activation loop in canonical kinases, and a fully assembled R spine is usually a prerequisite for kinase activity [16,30,31]. Functional prediction based on sequences – exceptions to the rule The aforementioned conserved residues have been used to predict nt-binding ability and/or catalytic activity of unknown PKs. The presence of an intact Gly-rich loop and VAIK motifs, for example, seem to have been the best predictors for catalytic activity in the past [32] – even in noncanonical active kinases like CASK, which lacks Asp184 but has an intact Gly-rich loop and a β3lysine. However, nt-binding ability and mechanisms cannot be reliably predicted from sequence data alone, as is shown by the innovative non-canonical nt-binding modes employed by several pseudokinases (see below). Thus, accurate biochemical and biophysical measurements that allow careful differentiation between nt binding, catalytic activity, and their possible physiological roles, are a necessity when analysing divergent proteins like pseudokinases. METHODOLOGY OF MEASURING NT BINDING The inherent properties of pseudokinases, i.e. low or absent kinase activity coupled to unknown nt binding, are often incompatible with traditional kinase assays and often multiple methods and rigorous controls need to be employed in order to obtain reliable results. Current techniques, and their challenges with respect to pseudokinases have been recently described in the excellent review by Lucet et al. [33]. In order to provide the background for the following inspection of nt-binding properties in pseudokinases, we will highlight some important technical aspects in the present study as well. Phosphoryl transfer or ATP hydrolysis activity of pseudokinases is generally orders of magnitude lower compared with active kinases, and therefore even tiny kinase contaminations can lead to false conclusion of pseudokinase catalytic activity [34]. As an example, in vertebrate mitotic checkpoint protein BUBR1 kinase-inactivating mutations and deletion studies demonstrated that the initially observed kinase activity was actually derived from contaminating kinases [35]. Furthermore, as with conventional kinases, choice of substrate can be critical, and the lack of phosphorylation of apriorilikely substrate candidates or an artificial substrate does not preclude that the protein could have .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 3 H.M. Hammar´ en, A.T. Virtanen and O. Silvennoinen Figure 2 Diverse ATP-binding pockets and nt-binding modes among pseudokinases Crystal structures of ATP-binding pockets of selected representative (pseudo)kinases and PKL proteins with varying nt-binding modes. Shown are human PKA (PDB: 4WB5), human RNase L (4OAV), human JAK2 JH2 (4FVQ), Arabidopsis thaliana BSK8 (4I94), human STRADα(3GNI), human VRK3 (2JII), A. thaliana BIR2 (4L68), Rattus norvegicus WNK1 (4Q2A), human MLKL (4MWI), human ROR2 (4GT4), human TRIB1 (5CEM) and human ADCK3 (4PED). ATP shown in WNK1, MLKL and ADCK3 was modelled based on PKA (4WB5), as no ATP-bound structures exist, even though they verifiably bind .......................................................................................................................................................................................................................................................................................................................................................................... 4c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. Nucleotide binding in pseudokinases specific activity in the context of a physiological protein complex [8,12]. X-ray crystallography gives atomic-level information on the structure of a nt-binding pocket and can be used to predict, or in the case of nt-bound structures, unequivocally verify nt binding. KSR2 [4], HER3 [5,36], TYK2 JH2 [37], JAK2 JH2 [38], STE20related adaptor alpha (STRADα)[39], ILK [40]andCASK[7], as well as a few other pseudokinases and PKLs from human and other species, have been crystallized in complex with nts providing solid proof and mechanistic information of nt binding (see Table 1). While X-ray crystallography is limited to determination of rigid protein structures, NMR is applicable for small proteins (Mr⩽30–40 kDa [41]) in a soluble state to reveal dynamic structures [42], and e.g. verify absence or presence of nt binding [43]. Assays measuring nt binding are often less sensitive for low levels of contaminations than kinase assays. Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are quantitative, label-free methods for detection of nt binding. ITC is the only method for direct measurement of thermodynamic parameters including enthalpy, Kdand stoichiometry for ligand– protein interaction, as it directly measures the absorbed or emitted heat during a (bio)molecular interaction [34]. SPR, on the other hand, provides direct information about binding kinetics and affinity through an optical assay [37,44]. Affinity (and to a lesser extent kinetics) estimates, can also be obtained using spectrofluorometric assays using intrinsic fluorescence measurements or fluorescently tagged nts. The intrinsic fluorescence method depends on tryptophan, tyrosine and phenylalanine residue(s), whose spectroscopic characteristics change in response to nt binding, which makes these assays somewhat limited in their applicability. More widelyused fluorescence applications use fluorescent nt analogues in fluorescence spectroscopy or fluorescence polarization assays. Mant (2(3)-O-(N-methylanthraniloyl)) [5,6,45]andTNP(2 -3- O-(2,4,6-trinitrophenyl)) [7,46] are commonly used labels, that show enhancement in fluorescence once bound to the hydrophobic ATP-binding pocket of a (pseudo)kinase. Fluorescence polarization (also known as fluorescence anisotropy) assays, using e.g. the long-wavelength fluorescently-labelled BODIPY– ATP-γ-S nt analogue, measure the increase in fluorescence polarization upon binding of a labelled small-molecule ligand to a protein. Due to its sensitivity, the assay is well-suited for screening purposes, but it has also been used to assess binding of nt analogues to pseudokinases [47]. Methods using tagged nts allow estimation of the Kdfor the nt analogues, but non-specific binding is possible. Values for unlabelled nts can be estimated by their ability to displace the fluorescent analogues, which is important, as the presence of fluorescent tags can change the binding properties of a nt [39]. For competition experiments the measurement window of the assay is, however, inherently restricted by the binding affinity of the probe in question. Binding of nts (or indeed any ligand) to proteins can also be analysed using thermal shift assays (TSA), which measure changes in thermal stability of a protein upon binding of ligands. Changes in thermal stability are measured by observing heatinduced unfolding of the protein using, e.g. intrinsic fluorescence of a protein [24,34] or extrinsic fluorescence of a dye [9]asa readout. Ligand binding generally stabilizes protein structures, and results in an increased melting temperature (Tm). Correlation between Tmand Kdor IC50 is dependent on the protein–ligand pair in question, and TSAs should not be used as a sole method for Kddetermination [48,49]. Furthermore, for low-affinity binders or cases where ligand binding does not cause large Tmshifts (Tm), sensitivity can be a problem. The methods described above are primarily suited for purified recombinant proteins. Nt affinity chromatography is one of the few tools for identification of nt binders also from cell lysates. The method uses affinity beads or immobilized ATP to capture nt-binding proteins and detection by, e.g. Western blotting [23] or mass spectrometry in a high-throughput approach [50]. In summary, estimates for nt-binding affinity between different methods can vary quite significantly, and the choice of method has to be often determined empirically. In general, label-free methods, like ITC, should be favoured for accuracy and precision, but due to limitations in, e.g. protein amount, other assays are also widely used. Furthermore, the functional role and relevance of nt binding can rarely be assessed using recombinant proteins alone, and usually requires site-directed mutagenesis in cellular assays. Additionally, the modes of nt binding vary widely in pseudokinases, and they can only be revealed by solving crystal or NMR structures. MODES OF NT BINDING IN PSEUDOKINASES Given the large variation of pseudokinase sequences especially at or near the nt-binding site (Figure 1), it is not surprising that pseudokinases display a wide variety of different nt-binding mechanisms. The mechanism of ATP binding is currently unknown in most pseudokinases, but structural information of 21 pseudokinases (Table 1) has shown that pseudokinases can bind Figure 2 Continued. adenine nts. ATP or ATP-analogues (e.g. AMP-PNP for BSK8) are shown as sticks with elements coloured as follows: carbon: black, oxygen: red, nitrogen: blue, phosphorus: orange. Divalent cations are shown as purple spheres. The halide ion in WNK1 is shown in green. The R spine is shown as a beige volume filling model, whereas the top of the C spine, encompassing the hydrophobic purine-binding pocket is shown in light blue. Hydrophobic side chains occluding the purine-binding pocket are shown as part of the C spine for VRK3, BIR2 and ROR2, where the pocket is occluded. Gly-rich loop glycines are shown as grey spheres with Gly-rich loop side chains omitted, unless of special note. Water molecules from the crystal structures have been omitted for clarity. *Only one possible conformation given for Gln486, ADCK3 is shown. .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 5 H.M. Hammar´ en, A.T. Virtanen and O. Silvennoinen Table 1 Summary of published pseudokinase crystal structures Abbreviations: P-PCP, β,γ-methyleneadenosine 5-triphosphate; AMP-PN, AMP phosphoramidate. *Crystallized with surface mutations (W659A, W777A, F794H). †Inferred from biochemical data or close homology to published structures showing the nt-binding mode. ‡Can be made cation-dependent with four point mutations [83]. §Mutated as explained in [77]. ¶Not identical with ROP2 sequence in 2W1Z. Crystal structures Protein PDB ID Species Ligand Complex Kdfor ATP Binding mode Other KSR2 2Y4I Homo sapiens ATP +Mg MEK1 Unknown 1 cation Active, phosphorylates MEK1 [4] HER3 4OTW H. sapiens Bosutinib – 10−6M[5] 1 cation Active, autophosphorylates its own intracellular region, when immobilized on vesicles [5] 4RIW H. sapiens AMP-PNP + Mg EGFR KD (V624R, F973A, L977A) 4RIX (Q709R) H. sapiens AMP-PNP + Mg EGFR KD (V624R, F973A, L977A) 4RIY (E909G) H. sapiens AMP-PNP + Mg EGFR KD (V624R, F973A, L977A) 3KEX H. sapiens AMP-PNP + Mg – 3LMG H. sapiens AMP-PNP + Mg – TYK2 JH2 3ZON H. sapiens IKK1 – 10−5M[37] 1 cation Inactive [37] 4WOV H. sapiens BMS-066 – 4OLI H. sapiens Inhibitor 7012 JH2-JH1 (D1023N) 5C03 H. sapiens ATPγS+Mg – 5C01 H. sapiens Pyrazine inhibitor – JAK2 JH2 4FVP* H. sapiens –– 10 −6M[6,45] 1 cation Active, autophosphorylates on S523 and Y570 [6] 4FVQ* H. sapiens ATP +Mg – 4FVR (V617F)* H. sapiens ATP +Mg – JAK1 JH2 4L00 H. sapiens –– 10 −6M[45] 1 cation†Inactive [65] 4L01 (V658F) H. sapiens –– MLKL 4BTF Mus musculus –– 10 −5M (TSA) [26] No cation† [9,25,26] Inactive [25] 4MWI H. sapiens –– 4M67 H. sapiens –– 4M68 M. musculus –– 4M69 M. musculus –RIP3KD STRADα3GNI H. sapiens ATP MO25 10 −4–10−6M [39,82] No cation [9] Inactive [140] 2WTK H. sapiens AMP-PNP MO25 +LKB1 .......................................................................................................................................................................................................................................................................................................................................................................... 6c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. Nucleotide binding in pseudokinases Table 1 Continued. Crystal structures Protein PDB ID Species Ligand Complex Kdfor ATP Binding mode Other VRK3 2JII H. sapiens – – None N/A Inactive ILK 3KMW H. sapiens ATP +Mg α-Parvin 10−6M[34] 1 cation Inactive [34,40] 3KMU H. sapiens –α-Parvin 3REP H. sapiens ATP +Mn α-Parvin CASK 3C0G H. sapiens 3-AMP – 10−3M[7,9] No cation Active, cations inhibit binding and activity. [7,24,83] 3C0I H. sapiens 3-AMP – 3C0H H. sapiens AMP-PNP (only AMP visible) – 3TAC H. sapiens – Liprin-α2 3MFS (4M)‡H. sapiens AMP-PNP – 3MFU (4M)‡H. sapiens AMP-PNP + Mn – 3MFT (4M)‡H. sapiens –– 3MFR (4M)‡H. sapiens –– ROR2 3ZZW H. sapiens – – None [55] N/A Inactive [55,102] 4GT4 H. sapiens –– BIR2 4L68 Arabidopsis thaliana – – None [94] N/A Inactive [94] BSK8 4I92 A. thaliana – – Unknown 1 cation Inactive [76] 4I93 A. thaliana –– 4I94 A. thaliana AMP-PNP – Titin 4JNW H. sapiens –– 10 −4M[113] Unknown Disputed [112–114] 1TKI H. sapiens –– PAN3 4CYI Chaetomium thermophilum ATP +Mg – Unknown, but probably rather high affinity [78] 1 cation Unknown, physiological role of PKD is to shuttle polyribonucleotides to PAN2 [81] 4CYJ C. thermophilum ATP +Mg PAN2 4CZY Neurospora crassa AMP-PNP + Mg PAN2 4BWK N. crassa ATPγS– 4BWX§N. crassa ATPγS+Mg 4BWP Drosophila melanogaster AMP-PN – 4XR7 Saccharomyces cerevisiae – PAN2 ROP2 2W1Z T. gondii – – None [43] N/A Inactive [43] 3DZO¶T. gondii Mg – ROP5B 3Q5Z T. gondii – – Unknown 2 cations Inactive [110] 3Q60 T. gondii ATP +Mg – 4LV5 T. gondii ADP IRGa6 (M. musculus) .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 7 H.M. Hammar´ en, A.T. Virtanen and O. Silvennoinen Table 1 Continued. Crystal structures Protein PDB ID Species Ligand Complex Kdfor ATP Binding mode Other ROP5C 4LV8 T. gondii ADP +Mg IRGa6 (M. musculus) Unknown 1 cation Unknown ROP8 3BYV T. gondii Mg – Unknown Unknown Unknown RNase L 4O1O Sus scrofa –2-5A10 −3M[53] 2 cations Inactive [52–54] 4O1P S. scrofa AMP-PNP + Mg 2-5A 4OAU H. sapiens ADP +Mg 2-5A 4OAV H. sapiens AMP-PCP +Mg RNA +pUp WNK1 4Q2A Rattus norvegicus Br – Unknown Unknown Active [124] 3FPQ R. norvegicus –– 4PWN H. sapiens –– MviN 3OTV Mycobacterium tuberculosis – – None [47] Unknown Inactive [47] 3OUK M. tuberculosis –– 3OUN M. tuberculosis – FhaA 3UQC M. tuberculosis –– ADCK3 4PED H. sapiens – – Binds preferentially ADP [108] Unknown Inactive, can be activated with a single Gly-rich loop mutation [108] ATP in a canonical fashion employing two cations or, as in most cases, in a non-canonical fashion with one or no cations visible in the crystal structure. In addition, some pseudokinases do not bind nts at all, and obtain either an active or inactive conformation by amino acid substitutions and structural rearrangements. Finally, a recent analysis of 31 pseudokinases showed that some pseudokinases were stabilized by the presence of only cations without nts [9], but the significance of this finding is currently unclear. In the present study, we provide a comprehensive structural view on nt binding in pseudokinases with a focus on the nt-binding mode in pseudokinases for which structural data are currently available. Bind nts PKA PKA represent the archetype for a canonical PK [51], and its mode of nt binding is the most prevalent among known PKL members. PKA binds its nt ligand ATP with two divalent cations positioned between the phosphates using the critical residues described above (Figure 2). RNase L The only known pseudokinase to date displaying a canonical nt-binding mode is the mammalian endoribonuclease RNase L. RNase L functions in the type I interferon (IFN) response where it is activated by 2,5-oligoadenylate (2-5A) second messengers and cleaves intracellular RNA. RNase L consists of an ankyrin repeat (ANK), a PK and a RNase domain. Despite its initial classification as an active kinase [1], the current consensus is that RNase L is an inactive pseudokinase [52–54], as it does not autophosphorylate [52], or phosphorylate the generic substrate myelin basic protein (MBP) [54]. The PK in RNase L is thought to function as a scaffold for homodimerization [54]. RNase L binds ATP with micromolar affinity and ATP binding activates RNase activity both in vitro and in cells, but is dispensable for RNase L dimerization in the presence of 2-5A [53]. The two recently published crystal structures of human and porcine nt-bound RNase L [53,54] show a PK fold with complete R and C spines resembling the traditional active kinase conformation (Figure 2)[31]. The structures, however, also show a non-canonical C lobe with substitutions in the activation loop and substrate recognition sites, which probably account for the inactivity of RNase L [52]. Both solved structures show a fully .......................................................................................................................................................................................................................................................................................................................................................................... 8c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. Nucleotide binding in pseudokinases ordered nt in the ATP-binding pocket, which resembles RNase L’s closest active homologue inositol-requiring protein 1 (IRE1). Nt binding is coordinated by conserved PK residues, except one uncommon substitution at Gly186 (Asp505, RNase L), which forms an additional interaction to one of the cations (Figure 2). The binding pocket is also flanked by the structured PK-ANK linker, which participates in 2-5A binding and interdomain interactions [53,54]. HER3 The most common nt-binding mode among the known pseudokinase structures is a one-cation binding mode first seen in human epidermal growth factor family pseudokinase HER3 (also known as ERBB3). As with other kinase families, pseudokinases can also be found in the group of 58 human receptor tyrosine kinases (RTKs), eight of which have been suggested to contain intracellular pseudokinase domains (PKDs) [55]. Clinically the most prominent of these is HER3, which mediates cellular signalling through ligand-induced heterodimerization with epidermal growth factor receptor (EGFR) and HER2 and allosteric activation of their kinase activity [36]. Its clinical relevance is exacerbated by the fact that HER3-mediated HER2activation can cause resistance to HER2-targeting cancer therapies [56]. The most noticeable features of the kinase domain of HER3 are the lack of Glu91 (substituted by His740, HER3, numbering as in PDB: 4RIW) and Asp166 (Asn815, HER3), the latter of which leading to the classification of HER as a pseudokinase [1,3]. The domain is, however, able to bind ATP tightly with a Kdof ∼1μM[5], and several crystal structures with the ATPanalogue adenylate-imidodiphosphate (AMP-PNP) have been reported (Table 1)[5,36,57], all of which show nt-bound HER3 in an inactive αC-out conformation. The nt binds together with a single Mg2+cation coordinated between the AMP-PNP phosphates, Asn171 (Asn820, HER3)andAsp 184 (Asp833, HER3). The conserved β3 lysine (Lys723, HER3) binds to both the αphosphate and Asp833, HER3 in a non-canonical manner most likely due to the lack of a Glu91-equivalent in αC. Interestingly, HER3 shows catalytic trans-autophosphorylation activity in vitro, when immobilized on vesicles at high local concentrations [5]. The biological relevance of this is uncertain, however [10,55], as the weak kinase activity has been reported to be dispensable at least for HER3-mediated ligand-dependent signalling [55]. JAK JH2 Janus kinases (JAKs) are non-RTKs with a rare domain architecture containing a pseudokinase (JAK homology 2, JH2) and a kinase domain (JH1) in tandem. JH2 mediates important regulatory functions and is needed both for suppression and full activation of JH1 activity [58–62]. JAKs function in a myriad of critical biological processes ranging from regulation of the immune system to haematopoiesis and metabolism [63]. The identification of multiple disease driver mutations in JAK JH2s have made them probably the most clinically studied group of pseudokinases to date. The most prominent of these mutations is the V617F mutation in JAK2 JH2 underlying multiple myeloproliferative neoplasms [64]. While JAK2 JH2 has been shown to possess low autoregulatory kinase activity [6], JAK1 and TYK2 JH2s are probably catalytically inactive pseudokinases as no autophosphorylation or kinase activity towards exogenous substrates has been detected [37,65]. Three of the four JAK JH2 domain structures have been solved over the last few years. The JH2s in all three (JAK1, JAK2, TYK2) are very similar and show a kinase domain structure with an accessible nt-binding site, a partially degraded Gly-rich loop (Gly50 and Gly52 are present, however) and an abnormally short activation loop [37,38,65]. The structures resemble the HER3 PKD in their nt-binding site architecture, as the canonical Lys72–Glu91 bond is replaced with a Lys72–Asp184 bond and an asparagine substitutes for Asp166 in the catalytic loop (Figure 1). In JAK2 JH2, for example, the nt phosphates coordinate around one manganese cation bound mainly by the conserved Asn171 (Asn678, JAK2). The phosphates bind between Lys72 (Lys581, JAK2), the Gly-rich loop backbone at Ser53 (Thr555, JAK2), as well as Lys677, JAK2, which performs the same role as Lys168 but is located two residues downstream along the catalytic loop (Figures 1Band2). Lys677, JAK2 is a lysine or arginine in all JAK JH2s and has been shown to be essential for ATP binding in JAK2 JH2 [45]. This resembles tyrosine kinases, which use an arginine at this position to substitute for Lys168 [66]. The hydrophobic lining of the purine-binding pocket is conserved in all JAK JH2s, including a non-canonically large aliphatic residue in β3 (Leu579, JAK2). JAK2 and JAK1 JH2’s bind ATP with micromolar affinity [6,45], whereas the ATP-binding ability of TYK2 JH2 has been somewhat unclear. In their comprehensive TSA screen of pseudokinases, Murphy et al. [9], observed a slight positive Tm shift upon addition of 200 μM ATP to TYK2 JH2, whereas Tokarski et al. [67] did not observe signs of ATP binding in their TSA or competition assays. TYK2 JH2 has, however, (along with JAK1 JH2) been found to be able to bind multiple kinase inhibitor scaffolds with submicromolar affinity [67,68]. A recent crystal structure of adenosine 5-[γ-thio]triphosphate (ATPγS)-bound TYK2 JH2 confirms that the domain is indeed capable of binding ATP. Furthermore, MANT-ATP, SPR and TSA experiments showed that the domain binds ATP with a Kd∼15–20 μM[37]. JAK2 JH2 has been found to possess low catalytic activity that phosphorylates two regulatory residues (S523, Y570) in JAK2 [6]. Phosphorylation of these residues facilitates the autoinhibitory interaction between JH1 and JH2 [69,70]. JAK1 and TYK2, however, do not possess kinase activity, despite binding ATP [37,65]. Comparison of the three JH2 structures shows that they all contain a helix (αAL) in the activation loop that in JAK1 and TYK2, but not in JAK2, is stabilized by two salt bridges and may block the substrate entrance [37]. Furthermore, S523 and Y570 residues are not conserved in JAK1 and TYK2 and the lack of suitable substrates may also account for the lack of phosphorylation-mediated regulation that may represent an ancestral function in JAK JH2 (JAK2 is the ancestral form). Crystal structures of apo and ATP-bound forms do not show major binding-induced changes in TYK2 JH2 [37], and for JAK2 .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 9 H.M. Hammar´ en, A.T. Virtanen and O. Silvennoinen concentrations are in the millimolar range [132], so nt binding should still occur in a physiological milieu even at relatively low affinities. Some very low affinity ATP binders (e.g. TRIB2, CASK) have even been hypothesized to function as cellular nt concentration sensors [24]. Functional ATP-binding pockets are required for the physiological function of multiple pseudokinases, even in the absence of catalytic activity. The detailed structural consequences of nt occupation, however, are in many cases not obvious even when using multiple biophysical methods (e.g. ILK [34]) or when crystallographic structural data with and without nt are available (see, e.g. JAK2 JH2 [45]). In these cases methods able to detect subtle changes in protein dynamics (e.g. NMR or computational methods) could provide new insights. Nevertheless, in most cases structure-guided, targeted, carefully controlled mutagenesis studies, complemented where possible with a chemical genetics approach [133], are the key to answering questions of importance of nt-binding pocket occupation. Pseudokinases play an important regulatory role in cellular signalling, and abnormal function of several human pseudokinases has been associated with human diseases [12]. The most prominent examples of this are probably the JAK2 JH2 V617F mutation underlying multiple myeloproliferative neoplasms [64], and HER3 overexpression in many cancers [134]. In total, over 60 mutations in the human pseudokinome have been shown to cause, or be linked to, various malignancies [11–13]. With its direct link to pseudokinase regulatory functions, the nt-binding site of many pseudokinases poses an interesting option for pharmacological interventions. For example, the E351K mutation in MLKL increases affinity for ATP and is associated with human lung carcinoma [26], and mutations in the ATP-binding pocket of JAK JH2 revert the hyperactivation of pathogenic JAK JH2 mutants [45]. To date, only a few pseudokinases, namely HER3 [135], MLKL [136] and TYK2 [67], have been attempted to target pharmacologically, providing important proof-ofprinciple results that targeting pseudokinases could be a viable therapeutic option. The results of pharmacological targeting of MLKL and TYK2 JH2 are significant and encouraging, but it should be kept in mind that the functions of pseudokinases are very diverse and not as directly predictable as for active kinases. 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