Monolith instrument

ImuA was labeled using the His‐tag Protein Labeling Kit RED‐Tris‐NTA 2nd generation (NanoTemper Technologies) according to the manufacturer's directions in the supplied MST buffer, using 200 nM ImuA (molar dye: protein ratio ≈1:4) at room temperature for 30 min in the dark. Unreacted dye was removed by centrifugation at 14,000×g for 10 min at 4°C.
Labeled ImuA was diluted to 100 nM with MST buffer (20 mM, 0.6 M NaCl, 10% v/v glycerol, 2 mM BME, 0.01% Triton‐X100). ImuB_NΔ34 was serially diluted 2‐fold in MST buffer containing 0.01% Triton‐X100, with the highest concentration starting at 54 μM. For the MST measurement, each ImuB_NΔ34 sample was mixed with equal volumes of ImuA, resulting in final concentrations of 50 nM ImuA and ImuB_NΔ34 ranging from 27 μM to 0.82 nM and incubated for 20 min. at room temperature. Samples were then loaded into Monolith capillaries (NanoTemper Technologies) and placed in the Monolith instrument (Nanotemper Technologies) for MST measurements at 25°C. 8% LED power and medium MST power were used in the data collection. Three independently pipetted measurements were analyzed from an MST‐on time of 21 s with the MO. Affinity Analysis software (version 2.3, NanoTemper Technologies).

HEK-293T cells (CL-0005) were purchased
from Procell Life
Science & Technology Co., Ltd. (Wuhan, China). Cells were transfected
with expression plasmids for pEGFP-C1 and pEGFP-C1-MrgprA3 (synthesized
by Sangon Biotech Shanghai, China), and the lysates were collected
as assay buffer for MST experiments. A NanoTemper Monolith instrument
(NT.115) (NanoTemper Technologies, München, Germany) was employed
for measuring thermophoresis. Samples were loaded into Monolith NT.115_Pico MST standard-treated capillaries and measured using Monolith
NT.115_Pico and MO.Control software (NanoTemper Technologies)
at 37 °C.