Molecular basis for the selective and ABA-independent inhibition of PP2CA by PYL13

Abstract

PYR1/PYL/RCAR family proteins (PYLs) are well-characterized abscisic acid (ABA) receptors. Among the 14 PYL members in Arabidopsis thaliana, PYL13 is ABA irresponsive and its function has remained elusive. Here, we show that PYL13 selectively inhibits the phosphatase activity of PP2CA independent of ABA. The crystal structure of PYL13-PP2CA complex, which was determined at 2.4 Å resolution, elucidates the molecular basis for the specific recognition between PP2CA and PYL13. In addition to the canonical interactions between PYLs and PP2Cs, an extra interface is identified involving an element in the vicinity of a previously uncharacterized CCCH zinc-finger (ZF) motif in PP2CA. Sequence blast identified another 56 ZF-containing PP2Cs, all of which are from plants. The structure also reveals the molecular determinants for the ABA irresponsiveness of PYL13. Finally, biochemical analysis suggests that PYL13 may hetero-oligomerize with PYL10. These two PYLs antagonize each other in their respective ABA-independent inhibitions of PP2Cs. The biochemical and structural studies provide important insights into the function of PYL13 in the stress response of plant and set up a foundation for future biotechnological applications of PYL13.

Abscisic acid sensor RCAR7/PYL13, specific regulator of protein phosphatase coreceptors

“…In two recent studies, RCAR7 has been claimed to selectively inhibit PP2CA independent of ABA and not to bind ABA because of changes in conserved amino acid residues involved in ABA binding (41,42). We found no evidence for this claim.…”

“…S2). Q38 of RCAR7 has been postulated to disrupt coordination of the carboxyl group of ABA mediated by a conserved lysine residue in other RCARs, and the bulky phenylalanine residue of RCAR7 at position 71 might generate a "steric clash" with the methyl group of ABA (41). RCAR7 Q38L , RCAR7…”

“…However, the low ABA sensitivity of the RCAR7-HAB1 S322P,Q386N complex implies additional structural constraints for efficient HAB1 regulation. The crystal structure of RCAR7-PP2CA revealed an additional interaction domain provided by a zinc-finger domain present in PP2CA and HAImember PP2Cs (41), which might provide an explanation for the high affinity of PP2CA in vitro and the high efficiency of RCAR7 to antagonize PP2CA and HAI2 action on ABA signaling. Several studies identified amino acid residues of RCARs (7,14,40,46) that alter interaction with PP2Cs, but none have reported so far on successful alteration of PP2C to establish RCAR binding.…”

Abscisic acid perception and signaling: structural mechanisms and applications

“…Reconstitution assays in Arabidopsis protoplasts showed that all PYL members except for PYL13, which is ABA-insensitive [110] and was therefore not tested, are able to activate ABA-induced gene expression [100] , suggesting functional overlap between the PYL members. In line with this notion, multiple mutations, such as pyr1pyl1pyl2pyl4 quadruple mutations [6] and pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutations [117] , are required to generate robust ABA-insensitive [117] .…”

“…The earliest PYL structural studies were based on PYR1, PYL1, and PYL2 [102][103][104][105][106] . To date, the structures of PYL3, PYL5, PYL9, PYL10, and PYL13 have also been solved, either in their apo or ligand-bound forms or in complexes with PP2C [107][108][109][110] . The currently available PYL structures represent more than half of the 14-member family of PYL proteins in Arabidopsis, and all of them exhibit the helix-grip fold, a hallmark of START domain/Bet v 1-fold proteins, which is characterized by a long C-terminal α-helix surrounded by a curved anti-parallel β-sheet and several smaller α-helices.…”