The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem-SH2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation which stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to doubly-phosphorylated tyrosine-containing motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the auto-inhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.
(Click on the small image to get a higher-resolution version.)
Figure 1 - Structure and function of the Syk family kinases.
A. Early B cell signaling events, left. Early T cell signaling events, right. In B cells and T cells a Src family kinase (yellow) phosphorylates ITAMs (phsophotyrosine denoted by yellow spheres) near the receptor. Binding of the tSH2 module to phosphorylated ITAMs recruits the kinase to the receptor. ZAP-70 signaling requires Lck, the Src family kinase, but Syk can initiate some signaling, even in the absence of Lyn, the B cell Src family kinase.
B. Domain architecture and autoinhibited structure of Syk family kinases (PDB: 4FL2, autoinhibited human Syk)
C. Activation of Syk family kinases by conformational change in the tSH2 module and phosphorylation. The tSH2 module binds to a doubly-phosphorylated ITAM (blue) and phosphorylation of tSH2-kinase linker stabilizes the open, active conformation.
Figure 2 - The crystal structure of the ITAM- free tSH2 module is similar to that of the ITAM-bound Syk tSH2 module.
A. The six molecules of ITAM-free Syk tSH2 aligned on the N-SH2 domain, showing the observed conformational heterogeneity.
B. Alignment on N-SH2 of all molecules in the ITAM-bound structure (PDB: 1A81, blue) and all in the ITAM-free structure (red). Only the C-SH2 is colored.
C. Left, alignment on the C-SH2 domain of the isolated, ITAM-free Syk tSH2 module (red) to that in the full-length auto-inhibited Syk structure (PDB:4FL2, white), resulting in a ~36 Â displacement of the N-SH2 domain of the ITAM-free tSH2 module with respect to its position in the auto-inhibited state. Right, the ITAM-free ZAP-70 tSH2 module (PDB: 1M61, blue) aligns well to the full-length auto-inhibited structure of ZAP-70 (PDB:4K2R, white), right.
Figure 3 - The Syk and ZAP-70 tSH2 modules show different deuterium uptake in and around the C-SH2.
Heat maps from representative peptides (residue numbers noted on the left) depicting differences in deuteration between the ITAM-bound and ITAM-free states of both tSH2 modules as measured by HDX-MS (n = 3). Red squares indicate increased deuterium exchange in the ITAM-bound state and blue squares indicate decreased deuterium uptake in the ITAM-bound state. A difference of at least ±0.5 Da was considered significant, and white squares indicate no change in deuterium uptake.
Figure 4 - The more flexible C-SH2 domain of ZAP-70 is rigidified by ITAM binding.
A. Deuteration difference heatmaps of all peptides, including different charge states, from the region spanning residues 161-192 in Syk (156-187 in ZAP-70). Peptides in ZAP-70 show a greater difference in deuteration between the ITAM-free and bound state.
B. Uptake plots from similar peptide in ZAP-70 and Syk. The peptides in Syk exchange similarly in the ITAM-free (dark blue for ZAP-70 and dark red for Syk) and bound states (light blue for ZAP-70 and light red for Syk). The corresponding peptides in ZAP-70 exchange similarly to Syk in the ITAM-bound state but are more flexible in the absence of peptide.
Figure 5 - The SH2 domains of the ITAM-free ZAP-70 move apart following the removal of the bound peptide.
The distance between the center of mass of the N- and C-SH2 domains in Syk tSH2 (red) and ZAP-70 tSH2 (blue). Simulations are smoothed over 10nsec. The histogram depicts the distribution of inter-SH2 distances sampled by either Syk (red) or ZAP-70 (blue).
Figure 6 - The differences between tSH2 modules are conserved in extant organisms.
A. Species tree generated based on the taxonomic relationships between organisms searched for Syk-family kinases.30 The occurrence of Syk-family kinases in each organisms is denoted on the bar graph below the tree. The occurrence of two Syk-family kinases in the genome corresponds to the emergence of jawed vertebrates (denoted by red circle). B. Alignment of only the tSH2 modules to either the human Syk or human ZAP-70 SH2 modules, from a representative organism from that order (common names: shark, lungfish, zebrafish, alligator, chicken, mouse).
Figure 7 - Sequence differences are conserved in both the Syk and ZAP-70 lineage.
The Armon conservation score at each position (Syk numbering) where the human Syk (red) and ZAP-70 (blue) sequence differ significantly. Overall ZAP-70 is more conserved than Syk, but there are positions which are conserved in only the Syk lineage or equally conserved in both lineages.
Figure 8 - Charged residues conserved in Syk but not ZAP-70 may contribute to increased stability of the C-SH2.
A. The residues in ZAP-70 with higher RMSF values than the corresponding regions of Syk are colored in green. Boxes highlight the C-SH2 domains of Syk (left) and ZAP-70 (right). In the Syk C-SH2 charged residues (red sticks) may be involved in stabilizing the fluctuations in this domain. D219 is able to interact with R217 and R197, forming cross β-strand interactions. These residues are not conserved in ZAP-70 (blue sticks). In Syk, R253 and D210 are positioned to interact.
B. Snapshots of the β-strands discussed in (A) at different points in the simulation. In both Syk and ZAP-70 these β-strands peel apart. The distance between two of the &beat;-strand residues is noted in the corner of each box and is depicted by the dashed line. The lower panel shows this distance throughout one of the simulations (plots of other simulations can be found in Supplemental Figure 2). The β-strands in ZAP-70 move apart to a greater extent and this separation is sustained for longer than in Syk.
C. All positions in the C-SH2 domain in which human ZAP-70 and human Syk differ are noted along the x-axis. Residues discussed in (A and B) are conserved in the Syk lineage but are not completely conserved in the ZAP-70 lineage, pointing to a potential constraint on this region during the evolution of Syk.
(Click on the small image to get a higher-resolution version.)
Supplemental Figure 1 - The ITAM-free tSH2 module of ZAP-70 is more dynamic than that of Syk.
(A) The C-SH2 domain of ZAP-70 and a section of the inter-SH2 linker are more dynamic than the corresponding regions in Syk as measured by the root-mean-square-fluctuation (Â) of each Cα atom in the individual domain about its average position in that domain across five independent simulations of each
of the ITAM-free tSH2 modules. Error bars represent the SEM (n=5).
Supplemental Figure 2 - Central β-strands in the C-SH2 of ZAP-70 fluctuate throughout the simulations.
The distance (Syk in red, ZAP-70 in blue) between two of the β-strand residues each pair of simulations. These β-strands in ZAP-70 move apart to a greater extent and this separation is sustained for longer than in Syk in most of the simulations.