A helix scaffold for the assembly of active protein kinases

Abstract

Structures of set of serine-threonine and tyrosine kinases were investigated by the recently developed bioinformatics tool Local Spatial Patterns (LSP) alignment. We report a set of conserved motifs comprised mostly of hydrophobic residues. These residues are scattered throughout the protein sequence and thus were not previously detected by traditional methods. These motifs traverse the conserved protein kinase core and play integrating and regulatory roles. They are anchored to the F-helix, which acts as an organizing “hub” providing precise positioning of the key catalytic and regulatory elements. Consideration of these discovered structures helps to explain previously inexplicable results.

Protein kinases represent a large protein superfamily that regulates numerous processes in living cells (1). Malfunction of this regulation typically leads to various diseases, including immunodeficiencies, cancers, and endocrine disorders (2). Multiple sequence alignment identified the most conserved motifs and defined universal subdomains in protein kinases (3). Solving crystal structures of different protein kinases demonstrated not only a conserved core but also the exceptional flexibility of protein kinases. This indicated an important role of dynamics and plasticity for this family (4, 5). Substantial progress has been made in understanding the regulatory mechanisms, although many questions still remain unanswered (6). Recently, we reported a new bioinformatics method that is capable of detecting conserved patterns formed by residues in space without any relation to protein sequence or main chain geometry. Originally, it was created for comparison of protein surfaces (7, 8), but later the method was expanded for analysis of the whole molecule and was termed “Local Spatial Patterns (LSP) alignment” (9). Application of the method to a set of serine/threonine and tyrosine kinases led to the discovery of an unusual structure, which we termed a “spine” (8). The most remarkable feature of the spine is that it is assembled during the protein kinase activation process and provides coordinated movement of the two kinase lobes. In deactivated kinases, the spine is usually broken because of the rearrangement of the C-helix and/or activation loop. Disassembly of the spine leads to general destabilization of the kinase molecule, which was previously observed in hydrogen–deuterium exchange studies (10, 11) and MD simulations (12). It was demonstrated subsequently that mutation of the spine residues leads to increased flexibility of the activation loop in MAP kinase ERK2 (13) and to a total inactivation of p38α MAP kinase (14).

Despite the fact that the spine is a conserved feature, present in all active eukaryotic protein kinases, it was not detected earlier as a conserved spatial motif. This is due, in part, to the highly unusual nature of its formation. It is comprised of four single residues coming from four different subdomains of the protein kinase molecule (III, IV, VIb and VII)^¶, which do not form a sequence “motif” in a traditional sense. 3D alignment of different kinases was also incapable of detecting this ensemble, because it does not form any contiguous main-chain pattern. This discovery drew our attention to the internal structure of the kinase catalytic core. Quite often, it is considered as an amorphous clustering of hydrophobic residues, a result of hydrophobic collapse in the protein folding process. However, it was shown that large ensembles of residues can be formed inside proteins, thereby creating allosteric signaling pathways (15, 16). Residues in these formations are precisely positioned, and their mutation abolishes the allosteric signal propagation. Existence of the hydrophobic spine demonstrated that such unconventional structures not only may be a part of allosteric signaling systems but also can perform structural and/or regulatory functions. Detection of such ensembles is not a trivial task, because they can be formed by residues that come from different parts of the polypeptide chain and do not form any motifs in terms of sequence or secondary/tertiary structure. Usually it requires a complicated multiple sequence alignment of hundreds or even thousands of sequences to achieve statistical equilibrium (15). In contrast, the LSP alignment does not need any sequence alignment, although it does require knowledge of the 3D structures. Its advantage, however, is that comparison of only two structures may provide meaningful insight into protein structure and function. The reason is that the LSP alignment not only detects the conserved patterns of residues but also ranks these residues according to their involvement in the patterns.

In our previous work, we analyzed only water-accessible residues (8). In this study, we used the LSP alignment to compare whole molecules of serine/threonine and tyrosine kinases. Inside the hydrophobic core of the kinases, we detected conserved unconventional motifs, similar to the spine reported earlier. We show that the F-helix, which is positioned in the middle of the large lobe, plays an integrating role by anchoring many hydrophobic motifs. These motifs traverse the entire molecule and orchestrate the catalytic process. They also provide diverse mechanisms for regulation. We define a second “spine,” which we term a catalytic spine, because it traverses both lobes but is completed by the adenine ring of ATP. This is distinct from the previous spine that we now refer to as the regulatory spine. The R and C spines are anchored to the N and C termini of the F-helix, respectively. We furthermore demonstrate that the APE motif, which is bound to the F-helix, nucleates substrate binding and allostery. A discussion of several published works shows that newly defined structures can be helpful for understanding the intramolecular machinery of protein kinases and their interactions with other proteins.

Results

LSP Alignment of Different Serine/Threonine and Tyrosine Kinases.

The most important information produced by the LSP alignment is expressed by the so-called involvement score (IS). It is defined for every residue of the compared proteins. If the score equals zero, this residue is not a part of any conserved spatial pattern. The higher the IS value, the more this residue is involved in formation of the conserved patterns, and the higher the probability that it is important for protein functionality [see supporting information (SI) Text for detailed explanation of the concept]. Although in the previous work (8), we described the major changes related to protein kinase activation, in this work, we have analyzed only active kinases and considered all residues, not just solvent accessible residues, as was done previously. We compared protein kinase A (PKA) from the AGC group to eight kinases from four groups: AGC (PKC and ROCK1), CMGC (CDK2 and SKY1), CaMK group (PHK and DAPK), and protein tyrosine kinases (SRC and IRK). All IS values obtained in eight comparisons are listed in the Table S1. Fig. 1 shows accumulated IS for each PKA residue.

Fig. 1.

LSP alignment of active conformation of PKA to active conformations of other kinase. Four different families were used in the comparison: AGC kinases (PKC and ROCK1), CMGC kinases (CDK2 and SKY1), calcium/calmodulin kinases (PHK and DAPK), and tyrosine kinases (SRC and IRK). Residues that constitute the two conserved spines are spread along the kinase sequence: R spine residues are marked as red dots; yellow dots mark the C spine residues. Highly scored αF-helix is marked by red square.

Newly detected residues with high IS were localized mostly in the hydrophobic core of the large lobe, predominantly in helices E and F, around the P + 1-loop and the APE motif (residues 201–210; here and subsequently, we will use mammalian PKA sequence numbering) (Fig. 1, Table S1). Several residues scored in every kinase were found in the H-helix. In addition, residues with exceptionally high IS, which were not appreciated previously, were detected in the N lobe and catalytic and activation loops: L¹⁰³, M¹²⁸, I¹⁵⁰, L¹⁷², and V¹⁸².

Defining Conserved Structural Motifs.

Traditionally conserved motifs are related to an amino acid sequence pattern (like DFG or APE motifs in protein kinases) or a combination of secondary structures (e.g., helix–turn–helix motif in DNA-binding proteins). However, as we showed earlier (8), proteins can contain unconventional structural motifs, which are not related to any sequence or secondary structure motifs. This raises important questions: what constitutes a conserved structural motif? Where is a border between two motifs? In the case of traditional motifs, the answer to the second question is intuitive; two different motifs have to be separated “far enough” from each other. However, in our case, when motifs are formed by amino acid residues coming from different parts of the primary sequence, this approach is irrelevant. In the current work, we have attempted to separate the large set of highly conserved residues, identified by the LSP alignment, into several different structural motifs based on their catalytic or regulatory roles.

For several reasons, we considered the F-helix as the central hub for these motifs. First, the IS level obtained by the F-helix residues was the highest through the entire molecule. Only the catalytic loop region together with the β₆ and β₇ strands was comparable. Second, it is positioned in the middle of the rigid hydrophobic core of the large C lobe, which means it is one of the most immobilized secondary structures in the molecule. Third, it is known that the F-helix serves as a signal-integrating element, which connects several key areas such as the substrate-binding residues and the catalytic loop (5, 17). These observations define the F-helix as a robust scaffold for the whole protein kinase molecule, where all of the motifs can be precisely positioned in space with respect to each other.

R Spine.

Earlier, we defined the regulatory spine as a motif of four hydrophobic residues, connecting the two lobes of the kinase (8). In the current work, we demonstrate that all structural motifs known to be important for catalysis are connected to the F-helix. In the case of the hydrophobic regulatory spine, this role is played by the aspartic acid D²²⁰ (Fig. 2), which is universally conserved in all eukaryotic and eukaryotic-like kinases (5, 17) and is the first F-helix residue with high IS. Further, we will refer to this five-residue motif (L⁹⁵, L¹⁰⁶, Y¹⁶⁴, F¹⁸⁵, and D²²⁰) as regulatory spine, or R spine.

Fig. 2.

R and C spines flank the F-helix and span the protein kinase core. The PKA structure is shown as a prototype. (A and B) The hydrophobic part of the R spine is shown as a red molecular surface. The C spine is colored yellow. The adenine ring of ATP completes the C spine. (C) Eight hydrophobic residues that constitute the C spine in 22 active kinases from six different groups (see Table S2 for the list of kinases).

C Spine.

The last highly scored residue at the C terminus of the F-helix was M²³¹ (Fig. 1). To the best of our knowledge, this residue was never considered as an important part of the kinase structure. However, our analysis shows that it starts a highly scored hydrophobic structure, which spans the large lobe and contacts the adenine moiety of ATP (Fig. 2). This structure includes two residues from the F-helix: L²²⁷and M²³¹; one residue from the D-helix, M¹²⁸; and the three hydrophobic residues, which comprise the β₇-strand: L¹⁷², L¹⁷³ and I¹⁷⁴. There are two highly ranked residues that contact the other side of the adenine ring: V⁵⁷ and A⁷⁰. Thus, in the presence of ATP, this ensemble connects the two lobes together, in a way similar to the R spine. By analogy, we termed this motif catalytic spine, or C spine. The major difference between these two spines is that the R spine formation depends on activation loop configuration, whereas the C spine depends on the presence of ATP. The C spine thus defines the adenine ring as a primary driving force for integration of the small and large lobes. As shown on the Fig. 2C, the C spine is conserved in both serine/threonine and tyrosine kinases. However, in more distant kinases, such as 3′,5′′-aminoglycoside phosphotransferase (APH) (18), this part of the molecule has a substantially different organization. It should be noted that the R spine, unlike the C spine, is present in APH (8). This suggests that the C spine is more sensitive to the nature of the substrate to be phosphorylated, whereas the R spine remains conserved in different kinases as soon as they have the conserved HRD and DFG motifs and the regulatory C-helix.

H-Helix Anchor.

There are three hydrophobic residues (W²²², I²²⁸, and Y²²⁹) and glycine (G²²⁵) from the middle part of the F-helix, which scored very high. These residues form a tight hydrophobic cluster with the four conserved leucines in the H-helix (L²⁶⁸, L²⁶⁹, L²⁷², and L²⁷³). As Fig. 3 shows, other residues from previously described structures are involved in the cluster: W²²², V²²⁶, and I²²⁸. An apparent function of the structure is to protect and stabilize the hydrophobic core around the F-helix. A characteristic feature of the structure is the position of L²⁷³, which is inserted into the void space between W²²² and V²²⁹, where a strictly conserved G²²⁵ is located. Such insertion is a characteristic of tetratricopeptide repeats (19), which provide a robust connection between two antiparallel α-helices. Most likely, the strong conservation of G²²⁵ is dictated by this feature.

Fig. 3.

αH-helix anchoring structure in PKA. Molecular surfaces of the highly scored residues are shown: olive, from the αF-helix, tan, from the αH-helix. L²⁷³ fits as a “knob” into a “hole” formed by conserved W²²², G²²⁵, and Y²²⁹.

Catalytic and Activation Loops Anchoring.

The maximal involvement score in our computation was received by another residue from the F-helix: A²²³. It was slightly higher than IS for such catalytically crucial aspartates as D¹⁶⁶ and D¹⁸⁴ (Fig. 1, Table S1). As shown in Fig. 4 A and B, A²²³ and neighboring L²²⁴ perform a very important function of anchoring L¹⁶⁷ from the catalytic loop. This leucine interacts both with R and C spines and provides exact positioning of D¹⁶⁶ and K¹⁶⁸ main chain (Fig. 4A). These residues are directly involved in phosphoryl transfer (20) and are universally conserved.

Fig. 4.

Catalytic and activation loop anchoring in PKA. (A) N- and C-terminal parts of the catalytic loop are secured by the spines. The middle part is docked to A²²³ and L²²⁴ from the αF-helix. Three catalytically active residues are shown: D¹⁶⁶, K¹⁶⁸, and N¹⁷¹. (B and C) Activation loop anchoring. Its N terminus binds to the β₈-strand that makes both polar and hydrophobic bonds to the β₇-strand from the C spine. The β₈-strand is also bound to the αF-helix via hydrophobic interaction with the conserved I¹⁵⁰. This positions the N terminus of catalytically active D184, whereas its C terminus is secured by the R spine.

L²²⁴ also serves as a basis for another highly scored residue from the E-helix: I¹⁵⁰ that defines the position of N terminus of the activation loop with the D¹⁸⁴ (Fig. 4 B and C). Stabilization of this aspartate is critical, because it binds a magnesium atom that bridges the β- and γ-phosphates of ATP. Such positioning is secured by a contact between I¹⁵⁰ and two hydrophobic residues from the β₈ strand: I¹⁸⁰ and V¹⁸². They interact with both spines and are a part of the β₇-β₈ sheet, a structural feature that is conserved in all protein kinases.

Substrate-Binding Structure.

A common feature for all protein kinases is that they phosphorylate polypeptide chains. This separates them from all other phosphotransferases and makes their substrate-binding structure substantially different (21). This structure is known to be formed by two major structural elements: the G-helix and the C-terminal part of the activation segment (5), referred to as the P + 1 loop (residues 198–205), where the substrate is bound to provide positioning of the phosphorylation site next to the γ-phosphate of ATP. Our analysis identified a set of highly scored residues in the subdomain VIII, with the conserved APE motif at the end of the activation segment. Residues from Y²⁰⁴ through E²⁰⁸ were detected in both serine/threonine and tyrosine kinases (Fig. 1, Table S1). Several residues received high scores only in serine/threonine kinases: T²⁰¹, E²⁰³, and I²⁰⁹. The major difference between serine/threonine and tyrosine kinases is that the size of tyrosine side chain is significantly larger. This explains the variation in geometry and sequence of the P + 1 Loop, where a hydrophobic residue following the phosphorylation site is accommodated (Fig. 5B).

Fig. 5.

Organization of the substrate-binding network in PKA. (A) V²²⁶ stabilizes Y²⁰⁴, which anchors the activation segment (colored bright red) and orients side chain of K¹⁶⁸. E²³⁰ interacts with the substrate arginine. (B) W²²² serves as a docking platform for the APE motif, which serves as a foundation for the P + 1 loop. (C) Closeup of the APE-motif interactions. R²⁸⁰ forms multiple hydrogen bonds to the αF-helix, the APE motif, and the αH-αI loop. (D) General chart of the YLAPEL motif that mediates interaction between the αF-helix, substrate binding, and docking sites.

The highest levels of IS were obtained by Y²⁰⁴ and A²⁰⁶, which are in direct contact with the highly scored residues from the F-helix: W²²² and V²²⁶ (Fig. 5B). This is consistent with our suggestion that all of the important structural motifs are connected to the F-helix. As we showed earlier, these two residues from the F-helix constitute a part of the rigid H-helix anchoring structure. Fig. 5 shows that they also play an important role as a foundation of the substrate-binding structure. V²²⁶ provides the exact positioning of the conserved tyrosine (Y²⁰⁴) in serine/threonine kinases (Fig. 5A) or tryptophan in tyrosine kinases. This tyrosine was reported to be an important allosteric “hot spot” in PKA (22–24). It is involved in hydrophobic interaction with the aliphatic part of K¹⁶⁸ from the catalytic loop and, together with another residue from the P + 1 loop (T²⁰¹), stabilizes the side chain of this catalytically important residue. W²²² tightly binds to the Ala-Pro pair from the APE-motif (Fig. 5 B and C) and nucleates the hydrophobic structure that positions four hydrophobic residues comprising the P + 1 loop (Fig. 5B). The APE-glutamate (E²⁰⁸) anchors the side chain of the highly conserved arginine from the loop between the H and I helices: R²⁸⁰ (Fig. 5C).

Discussion

During last two decades, protein kinases were studied intensively using sequence and structure alignments. Our recent work demonstrated that comparison of protein kinase surfaces can discover unconventional structural motifs that play an important regulatory role (8). Further development of the surface comparison method allowed us to include both water-exposed and buried residues in the analysis (9). The major objective of this work is to analyze conserved structural features in the hydrophobic core of different protein kinases. As we expected, all residues, which were known to be important for catalysis or for its regulation were highly scored, thus confirming the credibility of our method. Along with the well known residues, we discovered a set of residues that were not considered previously to be important elements of the protein kinase structure or function (Table S3). The major problem, however, was to separate the pool of the detected residues into meaningful structural elements. In the analysis, we followed a simple principle of functionality: there has to be a structure that coordinates the binding of ATP and a structure that coordinates the binding of the substrate. These structures have to be positioned with respect to each other in a precise and robust way. In our analysis, we found that the highest levels of the IS were obtained by the F-helix. Almost every residue of the helix was highly conserved and precisely positioned with respect to the other key residues responsible for catalysis, substrate binding, or regulation. Earlier, the helix was described as a signal integrating motif for the protein kinase molecule (5). In this work, we demonstrate that it serves as a central scaffold for active protein kinase assembly. Two conserved structures that flank the F-helix and span the protein kinase molecule are the most significant feature of the intramolecular architecture (Fig. 2). We termed these structures R and C spines. They represent unconventional structural motifs formed by residues coming from different parts of the protein sequence and do not form any sequential motifs in a traditional sense (Fig. 1). An important property of these structures is that they can be assembled and disassembled depending on the presence of ATP, the phosphorylation state of the activation loop, or the C-helix movement. This provides a significant conformational plasticity, which is very important for protein kinase functionality (4, 5). Furthermore, because the spines are comprised of hydrophobic residues, this makes the connection between different parts of the molecule firm but flexible. When the two spines are assembled, all catalytically important residues are securely positioned and ready for the catalysis (Fig. 4).

Substrate binding is also defined by residues from the F-helix: W²²², V²²⁶ and E²³⁰ (Fig. 5). The latter directly binds to the substrate and, for this reason, is relatively substrate specific. It was shown that presence of glutamate in this position is a characteristic of kinases with substrate preference for P-2 or P-5 arginines (25). These three residues position the C-terminal portion of the activation segment, which is usually associated with the APE-motif. Fig. 5D shows the general organization of the substrate binding elements. E²³⁰, together with V²²⁶, anchors Y²⁰⁴, which is highly conserved in serine/threonine kinases but substituted by tryptophan in tyrosine kinases. However, both tyrosine and tryptophan play a common role by providing a rigid platform for the N-terminal part of the substrate and by orienting the side chain of the catalytically active K¹⁶⁸ (arginine in tyrosine kinases) (Fig. 5A). W²²² serves as a docking site for binding alanine and proline in the conserved APE-motif (Fig. 5 B and D). The hydrophobic residue right before the alanine (L²⁰⁵ in PKA) defines the position of the P + 1 loop, which stabilizes the C-terminal part of the substrate (Fig. 5B). The main chain geometry of the six C-terminal residues of the activation segment is universally conserved through all serine/threonine and tyrosine kinases, even in casein kinase I (PDBID 2CHL), which does not have the APE-motif, W²²² or R²⁸⁰. This geometry is also precisely mimicked in the transphosphorylating protein kinases, when two interacting kinases exchange their activation segments (26). It emphasizes the crucial role of the YLAPEL-motif. Fig. 5D shows that it is related not only to substrate binding but also to docking sites on the protein kinase. Docking site A is located between the activation segment and the G-helix. It is a major binding site between the catalytic subunit of PKA and the A-domain of its regulatory subunits (27, 28). It was shown that docking of the CDK-interacting protein phosphatase KAP to CDK2 also occurs on docking site A (29). The second site is located in the loop between the H and I helices. This was shown to be an important interface in the PKA type RII holoenzyme, where the B-domain of the regulatory subunit binds to the catalytic subunit (28). One can suggest that, because of the proximity of this area to the substrate binding site and the universally conserved interaction between the APE-glutamate and R²⁸⁰, docking site B is important for other protein kinases as well.

Thus, we defined several conserved spatial structures in the hydrophobic core of protein kinases, which position the following three major elements: the peptide substrate, ATP, and the set of residues directly involved in the catalysis. Definition of the structures and their roles helps to interpret previously inexplicable results. For example, it was not clear why the E²³⁰Q mutant of PKA crystallized in an open apo-form, despite the fact that ATP was present in the solution (30). E²³⁰ is usually considered a part of substrate-binding complex, and it was not clear how it could influence ATP binding. However, a close look at the E²³⁰Q mutant structure shows that such a glutamate–glutamine substitute significantly destabilizes R¹³³ from the D-helix, which is bonded to the E²³⁰ in wild-type PKA. The arginine side chain in the E²³⁰Q mutant was not resolved, and the whole D-helix had elevated temperature factors. According to our classification, the D-helix constitutes the middle part of the C spine, which is responsible, in particular, for ATP binding (Fig. 2). Destabilization of the C spine foundation would explain the observed instability of ATP binding. This would lead to inability of the mutant to complete the C spine formation, promoting the apo-configuration.

A similar effect of the C spine destabilization was observed in the F³¹⁴A mutant of PKA (31). This phenylalanine is located in the C-terminal tail and contacts H¹³¹ and I¹³⁵ from the D-helix. Clearly, the phenylalanine-to-alanine mutation had to destabilize both the C-terminal tail and the C spine. Indeed, this mutation led to a significant decrease in thermal stability, a moderate decrease in affinity for ATP, and a nearly 20-fold decrease in the catalytic activity. Mutation of the neighboring residue I³¹⁵, which is not in contact with the D-helix, and thus the C spine, decreased thermal stability of the mutant and affinity for ATP and Kemptide but did not decrease activity. This indicates that stability of the C spine is important for optimization of the catalytic process.

Interconnectivity of the conserved spatial structures sheds light on long-range communication within kinase molecules, which has been observed by numerous authors (13, 14, 22, 24). The unphosphorylated apo-structures are typically the most disorganized: both the R and C spines are broken, and movements of the lobes are not coordinated. R spine assembly induced by phosphorylation or interaction with other activating entities such as cyclin for cyclin-dependent kinases causes significant ordering of the kinase core structure (11, 12). ATP binding completes the C spine formation and makes the molecule even more compact and primed for catalysis. Finally, substrate binding connects all parts of the molecule. The phosphorylation site is in the central cross-point for all conserved structures, and residues positioned close to it can substantially influence the intramolecular connectivity and thus, communication. A well examined case of such influence is Y²⁰⁴ from the YLAPEL-motif. Multiple studies demonstrated that mutation of this tyrosine to alanine decreased substrate binding and enzyme activity (22), caused destabilization in distal parts of the C lobe (23), and disrupted general intermolecular communication (24).

Methods

Modification of the Computational Method.

LSP alignment is a graph-theory-based method that compares two protein structures and detects similar spatial patterns made by residues in the proteins. The patterns are described by a pair of isomorphic graphs, where vertices correspond to the detected residues with edges connecting pairs of similar residues whose mutual orientation in space is conserved. Our previous work showed that functionally important residues are usually positioned in the middle of the graph with numerous connections to their neighbors. Alternatively, residues that play a supportive role are on a periphery of the graph with a fewer number of connections. The number of connections on the similarity graph was termed IS (see SI Text for detailed explanation of the involvement score concept). IS calculation in the current work was made according to the previously published algorithm (8) with a certain modification of similarity specification for residues. Earlier, we considered residues to be similar if their BLOSUM62 coefficient (32) was ≥2. However, this approach sometimes leads to suggestions that are not adequately justified. For example, valine and methionine are considered similar to isoleucine but not to leucine. A decrease of the threshold to 1 resolves this problem but, on the other hand, provides a rather doubtful suggestion that threonine can be substituted by proline, glycine, or aspartic acid. In the current work, we based our substitution matrix on one of the optimized substitution matrices (33) with consideration of the strong cysteine hydrophobicity (34) (see Table S4).

Protein Structures.

The following structures of protein kinases were used in the analysis: AGC kinases: PKA–PDBID: 2CPK; PKC–PDBID: 2JED; ROCK1–PDBID: 2ESM. CMGC kinases: CDK2–PDBID: 1FIN; SKY1–PDBID:1HOW. Calcium/calmodulin kinases: PHK–PDBID: 2PHK; DAPK–PDBID: 1JKK. Tyrosine kinases: IRK–PDBID: 1IR3; SRC–PDBID 1Y57.

Molecular graphics were prepared by using PyMOL (DeLano Scientific). Molecular surface was rendered with a probe radius of 1.4 Å.