Seemadri Subhadarshini
Published
1. Pseudokinases repurpose flexibility signatures associated with the protein kinase fold for non-catalytic roles
Protein kinases form an integral part of cellular signaling networks, phosphorylating target proteins and modulating their functional output. It is important to understand the sequence-structure-dynamics-function relations of protein kinases and their catalytically inactive counterparts, commonly referred to as “pseudokinases”. Previous work from the lab, reported the structural differences between protein kinases and pseudokinases. This works attempted to explore the similarities and differences in flexibilities of pseudokinases with respect to corresponding protein kinases by using normal mode analysis (NMA) and understand its functional implication.
Figure 1
The bilobal protein kinase-like fold in pseudokinases lack one or more catalytic residues, conserved in canonical protein kinases, and are considered enzymatically deficient. Tertiary structures of pseudokinases reveal that their loops topologically equivalent to activation segments of kinases adopt contracted configurations, which is typically extended in active conformation of kinases. Herein, Anisotropic Network Model (ANM) based Normal Mode Analysis (NMA) was conducted on 51 active conformation structures of protein kinases and 26 crystal structures of pseudokinases. Our observations indicate that although backbone fluctuation profiles are similar for individual kinase-pseudokinase families, low intensity mean square fluctuations in pseudo-activation segment and other sub-structures impart rigidity to pseudokinases. Analyses of collective motions from functional modes reveal that pseudokinases, compared to active kinases, undergo distinct conformational transitions using the same structural fold. All-atom NMA of protein kinase-pseudokinase pairs from each family, sharing high amino acid sequence identities, yielded distinct community clusters, partitioned by residues exhibiting highly correlated fluctuations. It appears that atomic fluctuations from equivalent activation segments guide community membership and network topologies for respective kinase and pseudokinase. Our findings indicate that such adaptations in backbone and side-chain fluctuations render pseudokinases competent for catalysis-independent roles.
Fig 1A depicts the canonical secondary structure organization (helices, strands and loops) of kinase-like fold. The regions corresponding to N-lobe, hinge and C-lobe are coloured in gray, blue and yellow, respectively, while those corresponding to catalytic loop (between β6-β7) and activation segment (encompassing β9, β10 and αEF) are indicated. In accordance, representative tertiary structures of protein kinase and pseudokinase are coloured (Fig 1B-G, left). Superimposed activation segments in all protein kinase structures in each family were in extended configuration, competent for substrate binding and catalysis (Fig. 1B, D, F (left); activation segments coloured in dark red). Topologically equivalent pseudo-activation segments superimposed in pseudokinase crystal structures, however, appeared coiled up and packed against the pseudoenzyme core (Fig. 1C, E, G (left); pseudo-activation segments coloured in dark green). This structural feature, common in pseudokinase structures in our dataset, was reminiscent of the inactive conformation of conventional kinases.
Figure 2. The nature of collective motions impacts large-scale fluctuations in kinases and pseudokinases. (A) Mean of collectivity values for first 25 non-zero normal modes in protein kinases (PK, red) and pseudokinases (PsK, green), related to RLK, TKL or TK families. (B) Maximum collectivity (κmax) derived from first 25 non-zero normal modes, for each PDB entry in the dataset, are plotted as a function of flexibility as calculated from area under the curve (AUC) of normalised square fluctuation profile for the mode displaying κmax (measured in arbitrary units). The shaded area for kinases (PK, top panel) and pseudokinases (PsK, bottom panel) indicates the range for flexibility which encompasses majority of data points. The markers for proteins closely related to RLK, TKL and TK family have been indicated. (C) Visual analysis of backbone fluctuations in selected case studies of RET kinase (PDB 6nja, top panel) and JAK2 pseudokinase (PDB 4fvq, bottom panel) crystal structures using Normal Mode Wizard. Tertiary structure representations of RET (top left) and JAK2 (bottom left) are colour coded to indicate N-lobe (gray), hinge (blue), C-lobe (yellow) and activation segments (red, PK; green, PsK). The backbone motions are represented by eigen vectors (shown as black arrows). Alongside, normalised square fluctuation profile for the mode displaying κmax, in RET (mode 2, top right) or JAK2 (mode 23, bottom right), is provided. Topologically equivalent structural elements of interest are marked.
Figure 2
Figure 3
Figure 3. Distinct community map architectures of kinase-pseudokinase pair based on inter-residue cross correlation matrices, which were further derived from all-atom normal mode analysis. Pairs of kinases and pseudokinases, sharing highest amino acid sequence identity in each family were chosen: RLK (BRI1, BIR2), TKL (BRAF, KSR2) and TK (EGFR, HER3). (A, C, E, G, I, K) Residue-by-residue cross-correlation matrices for BRI1 (A), BRAF (E) and EGFR (I) kinases as well as BIR2 (C), KSR2 (G), HER3 (K) pseudokinases are shown. For each matrix, colour-coded communities of residues exhibiting highly correlated motions (top X-axis) and secondary structure organisation (right Y-axis) are depicted. Boxes (dotted lines) correspond to β1 to β5 in N-lobe and FGHI helices in C-lobe. The colour scale (top of Fig. 3A) denotes cross-correlation values from −1.0 (blue) to 1.0 (red). Positive cross-correlations between structural elements of interest, as described in Section 3.4, are demarcated by coloured circles: black (A,C,E,G,I,K), red (A,C,E,G,I,K), green (C,G,K), yellow (A,E,I) and blue (A,E,I). (B, D, F, H, J, L) Coarse-grained community maps (left) of chosen kinases and pseudokinases, with the communities mapped onto their respective tertiary structures (right) are shown. The size of each community is proportional to the number of members (both residue backbones and side-chains) belonging to each community. The edges between communities depend on inter-community coupling. Communities with residues primarily belonging to N-lobe or FGHI helices in C-lobe are indicated with dotted circles or ellipses on the community maps (left). The community or communities that include the activation segment in kinases and pseudo-activation segment in pseudokinases are marked with asterisk (*).