A novel partially open state of SHP2 points to a "multiple gear" regulation mechanism

Abstract

The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.

Keywords: allosteric regulation; conformational change; fluorescence resonance energy transfer; single-molecule biophysics; tyrosine-protein phosphatase (tyrosine phosphatase).

Figure 1

Strategy of the single-molecule FRET assay.A, strategy for site-specific incorporation of Azido-p-Phe into SHP2 at positions Q87/K266. The engineered tRNA/aminoacyl-tRNA synthetase system introduces the Azido-p-Phe through specially recognizing the stop codon TAG at 87/266. Alkyne-Cy3 and Alkyne-Cy5 were conjunct to azido through the click reaction, in which THPTA was added to protect protein from denaturing. B, schematic of protein immobilization for smFRET measurements. 1D4-tagged Fluor-SHP2-87/266 was immobilized on the surface of passivated quartz slide via Fab-biotin. C, representative smFRET images of Fluor-WT-87/266, Fluor-E76A-87/266 were acquired with a prism-based TIRF microscope during donor excitation with a 532 nm laser. THPTA, Tris (3-hydroxypropyltriazolylmethyl) amine; smFRET, single-molecule fluorescence resonance energy transfer.

Figure 2

Single-molecule FRET assay reveals three conformational states of SHP2. A, (Left) FRET trajectories from individual WT-87/266 molecules were compiled into a population FRET histogram. Error bars are SEM. Decomposition of the FRET data resulted in three Gaussian curves representing the distribution of a low FRET state (red), medium FRET state (blue), and a high FRET state (black). Dashed lines indicate the distinct mean FRET values observed. The number (n) of smFRET traces compiled into each histogram is indicated. (Middle) real-time donor (green) and acceptor (red) intensity traces and FRET trace (blue) obtained from a single WT-87/266 molecule. Idealization of FRET trajectory (orange) was achieved by fitting to a three-state hidden Markov model. F, fluorescence; a.u., arbitrary units. (Right) TDPs for WT-87/266. Initial and final FRET (average FRET values before and after each transition respectively) were plotted as a two-dimensional chart between the three FRET states (scale at right). B, (Left) histogram of E76A-87/266. (Middle) representative smFRET trajectories for E76A-87/266 molecules. (Right) TDPs of E76A-87/266. C, the percentage of dynamic fractions of SHP2 WT and E76A. Error bars are SD, n = 3. Significance was determined by two-tailed t test. ∗∗p < 0.002. FRET, fluorescence resonance energy transfer; TDPs, transition-density plots.

Figure 3

Structural detail of the partially open state of SHP2 (E76A) revealed by MD simulation.A, (Left) predominant partially open conformations from structural cluster analysis are superimposed. The N-SH2 domain of the open, partially open, and closed states are colored in red, marine, black, respectively. The C-SH2 domain, PTP domain, and the catalytic loops in all states are colored in cyan, dirty violet, and orange. (Right) representative conformations indicate the motion of the N-SH2 domain from closed state to partially open and open states. The N-SH2 domains in the closed, partially open, and open states are colored in black, marine, and red, the other domains in three states are colored in gray. B, (Upper) the zoom in reviews of the closed, open conformations, and the superimposing of the predominant partially open conformations from the cluster analysis highlight the local arrangement of the N-SH2 domain in three states. (Bottom) cartoons for the rearrangement of the N-SH2 domain in the closed, partially open, and closed states. C–E, the three most predominant partially open conformations from the interaction-based classification. The N-SH2, the PTP domain, and the catalytic loops are colored in marine, dirty violet, and orange, respectively. C, hydrogen bonds and salt bridges are built between the residues in the D’E-loop (e.g., T59) and WPD-loop (G427), K-loop (R362) which restrict the motion of WPD-loop. D, hydrogen bonds and salt bridges between the D’E-loop (e.g., T59) and WPD-loop (G427), Q-loop (T507, E508), K-loop (R362) restrict the motion of WPD-loop and Q-loop. E, hydrogen bonds and salt bridges between the D’E-loop (e.g., G60) and K-loop (R362), Q-loop (T507, E508), P-loop (R465) restrict the motion of WPD-loop, P-loop, and Q-loop. MD, molecular dynamics.

Figure 4

Different mono-P segments shift the conformational equilibrium of SHP2 toward the partially open state.A, (Upper) cartoon for binding of mono-P segment EPIpYA-D to the N-SH2 domain of SHP2. (Bottom) histograms for E76A-87/266 in the presence of different concentrations of EPIpYA-D. B, (Upper) cartoon for binding of mono-P segment EPIpYA-C to the N-SH2 domain of SHP2. The sequence between EPIpYA-C and the binding pocket of the N-SH2 domain is mismatched. (Bottom) histograms for E76A-87/266 in the presence of different concentrations of EPIpYA-C. C, PTP activity of Azido-E76A-87/266 and Azido-E76A-87/266 in the presence of 0.25 μM EPIpY-D and EPIpYA-C. Significance was determined by two-tailed t test. ∗∗p < 0.002 Error bars are SD, n = 3. D, fraction of three states in the presence of 125 nM EPIpYA-D and EPIpYA-C, error bars are SEM, n = 3.

Figure 5

Conformational rearrangement in E76A-87/266 triggered by the bis-P segment pY1172-PEG₈-pY1222.A and B, (Upper) the cartoon for binding of mono-P segment pY1172 (a) and pY1222 (b) to SHP2. (Middle) histograms for E76A-87/266 in the presence of varying concentrations of pY1172 (a) and pY1222 (b). (Bottom) representative smFRET trajectories reveal transitions from low or high to medium FRET states in the presence of pY1172 (a) and pY1222 (b). C, (Upper) the cartoon for binding of the bis-P segment to SHP2. (Middle) histograms for E76A-87/266 in the presence of varying concentrations of pY1172-PEG₈-pY1222. (Bottom) a representative smFRET trajectory shows transitions from high to low FRET state through a medium FRET state in the presence of pY1172-PEG₈-pY1222. D, fraction of three states in the presence of 125 nM pY1172, pY1222 and pY1172-PEG8-pY1222. Error bars are SEM, n = 3. E, The PTP activity of apo Azido-E76A-87/266 and Azido-E76A-87/266 in the presence of 0.25 μM pY1172-PEG₈-pY1222, pY1172, and pY1222, respectively. Significance was determined by two-tailed t test. Error bars are SD, n = 3. smFRET, single-molecule fluorescence resonance energy transfer.

Figure 6

A “multi-gear” model for the regulation of SHP2 activity. The diagram represents the regulation of SHP2 by oncogenic mutations, allosteric inhibitors, and different activators. The binding of mono-P segment from receptors or docking proteins to a single (either N- or C-) SH2 domain switches SHP2 to the “low gear”, which semiactivates SHP2 with dominant partially open conformations. While, binding of bis-P segment to both SH2 domains can switch SHP2 to the “high gear”, which fully activates SHP2 with dominant open conformations. The oncogenic mutants can shift the conformational equilibrium of SHP2 from closed state to partially open and open states with hyperactivity, which can be retrieved by the addition of allosteric inhibitors.