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Sepuru, K. M.

Publications and source records attributed to Sepuru, K. M..

3 recordsLinked to original sources

Chemical genetics reveals cross-activation of plant developmental signaling by the immune peptide-receptor pathway

Cells sense and integrate multiple signals to coordinate development and defence. A receptor-kinase signaling pathway for plant stomatal development shares components with the immunity pathway. The mechanism ensuring their signal specificities remains unclear. Using chemical genetics, here we report the identification of a small molecule, kC9, that triggers excessive stomatal differentiation by inhibiting the canonical ERECTA receptor-kinase pathway. kC9 binds to and inhibits the downstream MAP kinase MPK6, perturbing its substrate interaction. Strikingly, activation of immune signaling by a bacterial flagellin peptide nullified kC9s effects on stomatal development. This cross-activation of stomatal development by immune signaling depends on the immune receptor FLS2 and occurs even in the absence of kC9 if the ERECTA-family receptor population becomes suboptimal. Furthermore, proliferating stomatal-lineage cells are vulnerable to the immune signal penetration. Our findings suggest that the signal specificity between development and immunity can be ensured by MAP Kinase homeostasis reflecting the availability of upstream receptors, thereby providing a novel view on signal specificity.

plant biology↗

Chemical inhibition of stomatal differentiation by perturbation of the master-regulatory bHLH heterodimer via an ACT-Like domain

Selective perturbation of protein interactions with chemical compounds enables dissection and control of developmental processes. Differentiation of stomata, cellular valves vital for plant growth and survival, is specified by the basic-helix-loop-helix (bHLH) heterodimers. Harnessing a new amination reaction, we here report a synthesis, derivatization, target identification, and mode of action of an atypical doubly-sulfonylated imidazolone, Stomidazolone, which triggers stomatal stem cell arrest. Our forward chemical genetics followed by biophysical analyses elucidated that Stomidazolone directly binds to the C-terminal ACT-Like (ACTL) domain of MUTE, a master regulator of stomatal differentiation, and perturbs its heterodimerization with a partner bHLH, SCREAM in vitro and in plant cells. On the other hand, Stomidazolone analogs that are biologically inactive did not bind to MUTE or disrupt the SCREAM-MUTE heterodimers. Guided by structural docking modeling, we rationally designed MUTE with reduced Stomidazolone binding. These engineered MUTE proteins are fully functional and confer Stomidazolone resistance in vivo. Our study identifies doubly-sulfonylated imidazolone as a direct inhibitor of the stomatal master regulator, further expanding the chemical space for perturbing bHLH-ACTL proteins to manipulate plant development.

plant biology↗

Long-range coupled motions underlie ligand recognition by a chemokine receptor

Chemokines are unusual class-A GPCR agonists because of their large size ([~]10 kDa) and binding at two distinct receptor sites: N-terminal domain (Site-I, unique to chemokines) and a groove defined by extracellular loop/transmembrane helices (Site-II, shared with all small molecule class-A ligands). Whereas binding at Site-II triggers receptor activation, the role of Site-I is not known. Structures and sequence analysis reveal that the receptor N-terminal domains (N-domains) are flexible and contain intrinsic disorder. Using a hybrid NMR-MD approach, we characterized the role of Site-I interactions for the CXCL8-CXCR1 pair. NMR data indicate that the CXCR1 N-domain becomes structured on binding and that the binding interface is extensive with 30% of CXCL8 residues participating in this initial interaction. MD simulations indicate that CXCL8 bound at Site-I undergoes extensive reorganization on engaging Site-II with several residues initially engaged at Site-I also engaging Site-II. We conclude that structural plasticity of Site-I interactions plays an active role in driving ligand recognition by a chemokine receptor.

biophysics↗