Structural insights into auxin recognition and efflux by Arabidopsis PIN1

Abstract

Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.

Chemical inhibition of PIN auxin transporters by the anti-inflammatory drug Naproxen

“…In line with the result that Naproxen has a higher binding affinity to PIN1 protein (with a Kd value of 5.83 µM, Fig. 3b) than the substrate IAA (with a Kd value of 186 µM, as revealed by SPR in Yang et al, 2022), suggesting that Naproxen may be a competitive inhibitor of IAA for PIN transporters. This complex structure aligns well with the NPA-bound structure of PIN1, with a root mean square deviation (r.m.s.d.)…”

“…To gain insight into the inhibition mechanism of Naproxen, we tried to determine the complex structure of PIN1 and Naproxen. Naproxen was added to the purified PIN1 protein bound with a synthetic nanobody, sybody-21 (Yang et al, 2022), prior to cryo-sample preparation. After cryo-EM data collection and processing, an EM map with an overall resolution of 3.7 Å was obtained (Fig.…”

“…These above physiological and cellular analyses suggest that Naproxen might be an auxin transport inhibitor. With advantage of an auxin efflux assay based on HEK293F cells and purified PIN1 protein (Yang et al, 2022), we tested if Naproxen targets PIN auxin transporters directly. In the auxin efflux assay, which measured residual 3 H-labelled radioactive IAA ([ 3 H]IAA) in cells after transfer into isotope-free buffer, we found that Naproxen treatment led to a higher retention level of [ 3 H]IAA in cells expressing PIN1 protein plus the activating kinase D6PK than the DMSO control, suggesting an inhibition of auxin export (Fig.…”

“…4e). However, the carboxyl group of NPA forms more hydrogen bonding with PIN1 like with the backbones of I582 and V583, and the benzene ring also forms hydrophobic stacking with Y145, L114 and V583 (Yang et al, 2022), which may contribute to the higher binding affinity with PIN1 than that of Naproxen.…”

“…Application of NPA to plants leads to various defects (e.g., interference of phototropism and gravitropism, inhibition of lateral root formation, suppression of vascular development, and deficiency in shoot development) due to suppression of auxin transport across cells. Recent biochemical and structural studies reveal that NPA directly binds to and inhibits the activity of PIN-FORMED (PIN) auxin transporters (Abas et al, 2021; Teale et al, 2021; Lam Ung et al, 2022; Su et al, 2022; Yang et al, 2022). Interestingly, NPA binds to the same pocket as the PIN substrate IAA, suggesting that NPA is a competitive inhibitor of PIN proteins, which is a paradigm shift in our understanding of NPA action.…”