Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases

Abstract

Bruton's tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

Fig 1. W395 is required for Btk activity.

(a) Btk linker-kinase domain structure (PDB ID: 3K54) showing the linker, N- and C-lobes, active site, and activation segment. (b) Key regulatory elements in the Btk linker-kinase domain are the R- and C-spines, orange and yellow, respectively. ATP completes the C-spine structure in the N-lobe but is omitted here for clarity. W395 is shown above the αC-helix and the residues in the conserved salt bridge, K430 and E445 are labeled. The C- and R-spines are supported by the αF-helix in the C-lobe of the kinase domain. (c) Initial velocity measurements comparing the activity of full-length Btk (domain structure is shown to the right) to full-length Btk (W395A) using the poly (4:1, Glu:Tyr) peptide substrate [11].

Fig 2. HDXMS reveals greater conformational sampling for active Btk linker-kinase domain.

(a) Constructs used for activity assays and HDXMS study. The underlined residue is vector derived and not part of the sequence of the Btk kinase domain. Both Btk linker-kinase and Btk (W395A) linker-kinase carried a hexahistidine tag (6His) at the C-terminus. (b) Western blot assay to probe phosphorylation of the Btk activation loop Y551 and Y783 in the PLCγ1 substrate. Anti-His antibody recognizes the 6-His tag on the Btk constructs and is used to detect the amount of Btk enzyme in each reaction. (c) Differences greater than 0.7 Da at any one of the five time points between 10 seconds and 4 hours in the hydrogen-deuterium exchange experiment are mapped onto two depictions of the Btk linker-kinase domain (PDB ID: 3GEN) and colored in blue. The side chain of W395 is red and labeled. (d-e) Deuterium exchange for peptides derived from the αC-helix and activation segment in Btk linker-kinase (red) and Btk (W395A) linker-kinase (blue). (f) Deuterium exchange for peptides derived from the linker and N-terminal region of Btk linker-kinase (red) and Btk (W395A) linker-kinase (blue). Complete HDX data is provided in Fig A in S1 Text. (g) Side-chain rotamer conformations of W395 in the structures of active (3K54) and inactive (3GEN) Btk linker-kinase.

Fig 3. MD simulations of Btk linker-kinase and Btk (W395A) linker-kinase domains.

Two replicate (200 nanosecond (ns) each) equilibrium simulations (black/light grey traces) are shown for Btk linker-kinase ((a), superimposed) and three 200 ns replicates of (W395A) linker-kinase domain are shown in (b,c,d). RMSD from the starting structure is reported for the backbone atoms (total), activation segment (540–547) and αC-helix (439–451). The distance (Å) between the side-chains of K430 and E445 is shown over the course of the simulations. The red dashed lines in each plot are included for ease of comparison between mutant and wild type trajectories. (e,f) Snapshots of structures from the wild type Btk linker-kinase simulation at 0 and 200 ns (e) and from the Btk (W395A) linker-kinase simulation at 0, 10 and 200 ns (f). The wild type Btk linker-kinase domain retains the active conformation throughout the 200 ns simulation. The K430/E445 salt bridge distance is indicated and the interaction between R544 and pY551 is evident at the beginning and end of the simulation. Btk (W395A) linker-kinase domain starts in the active conformation but moves toward the inactive state as early as 10 ns. Further transition to the inactive conformation (‘C-out’) is observed as the simulation progresses and at 200 ns the K430/E445 distance is 14.5Å and R544 contacts the side chain of E445 rather than pY551. F540 is shown at 0 and 200 ns in the Btk (W395A) linker-kinase structures to illustrate the shift from the active DFG conformation to inactive.

Fig 4. Principal Component Analysis.

(a,c) Percent variance captured by the first 10 PCs in Btk linker-kinase (a) and Btk (W395A) linker-kinase (c) domains. The red line is the cumulative variance captured by the PCs and the black line is the percentage of the variance captured by each individual PC. (b,d) Direction of motions in PC1, PC2 and PC3 for Btk linker-kinase (b) and Btk (W395A) linker-kinase (d) domain. Dotted lines show the axis of motion, the length of the vectors show the relative magnitudes and the arrowheads indicate the direction of motion.

Fig 5. Community analysis.

The community network in Btk linker-kinase (a) and Btk (W395A) linker-kinase (b) domains. (a,b left panel) The area of the circle indicates the number of residues within each community and the weight of the lines connecting communities is proportional to the extent of correlation between communities. (middle panel) Communities of residues mapped onto the Btk linker-kinase domain structure for Btk linker-kinase (a) and Btk (W395) linker-kinase (b). (right panel) R-spine communities for the Btk linker-kinase simulation (a) and the Btk (W395A) linker-kinase simulation (b).

Fig 6. Node-betweenness centrality index values reveals residues that bridge the R- and C-spines.

(a-c) Node-betweenness centrality index plot for Btk linker-kinase (a), Btk (W395A) linker-kinase domains (b) and Lck kinase domain (c). The threshold (dotted line) was set for Btk linker-kinase (a), such that 98.5% of the centrality index values are below the threshold value. The same threshold is used for Btk (W395A) linker kinase in (b) and Lck in (c). In (a) and (c) the centrality value for one residue, A428 in Btk and A271 in Lck, nearly reaches the threshold but was not included in our analysis since this residue is part of the previously defined C-spine in both kinases. (d) High centrality residues from (a) are mapped onto the structure of active Btk (3K54), labeled, and colored red. C-spine residues are yellow and R-spine residues are orange as in Fig 1B. (e) Spheres define the residues of the C-spine (yellow), R-spine (orange) and the newly identified bridging residues (red). The αF-helix is shown and ATP within the C-spine is depicted in stick form. The communities (Fig 5) of each of the four bridging residues are indicated. (f) High centrality residues in Lck (shown in (c)) are mapped onto the structure of the active Lck kinase domain (3LCK), labeled and colored red. As in (e) Lck C-spine residues are yellow and R-spine residues are orange, the αF-helix is shown and ATP is shown in stick form. I370 and L371 are high centrality residues in Lck (see (c)) and are part of the previously defined C-spine and therefore colored yellow.