Structural basis for receptor recognition of pollen tube attraction peptides

Abstract

Transportation of the immobile sperms directed by pollen tubes to the ovule-enclosed female gametophytes is important for plant sexual reproduction. The defensin-like (DEFL) cysteine-rich peptides (CRPs) LUREs play an essential role in pollen tube attraction to the ovule, though their receptors still remain controversial. Here we provide several lines of biochemical evidence showing that the extracellular domain of the leucine-rich repeat receptor kinase (LRR-RK) PRK6 from Arabidopsis thaliana directly interacts with AtLURE1 peptides. Structural study reveals that a C-terminal loop of the LRR domain (AtPRK6^LRR) is responsible for recognition of AtLURE1.2, mediated by a set of residues largely conserved among PRK6 homologs from Arabidopsis lyrata and Capsella rubella, supported by in vitro mutagenesis and semi-in-vivo pollen tube growth assays. Our study provides evidence showing that PRK6 functions as a receptor of the LURE peptides in A. thaliana and reveals a unique ligand recognition mechanism of LRR-RKs.

Fig. 1

AtPRK6^LRR specifically interacts with AtLURE1.2 in vitro. a AtLURE1.2 interacts with AtPRK6^LRR in a pull-down assay. The purified AtPRK^LRR and MIK1^LRR, MIK2^LRR proteins with 6 × His at the C-terminus bound to Ni-NTA were individually incubated with an excess of AtLURE1.2 protein. After extensive washing, the bound proteins were eluted and visualized by Coomassie blue staining following by SDS-PAGE. b AtLURE1.2 binding does not alter the monomeric state of AtPRK6^LRR in solution. Upper panel: gel filtration profiles of AtPRK6^LRR, AtLURE1.2, and AtPRK6^LRR-AtLURE1.2 complex. The black arrows indicate the molecular weights around the AtPRK6^LRR-AtLURE1.2 complex and AtLURE1.2. A₂₈₀ (mAU), micro-ultraviolet absorbance at the wavelength of 280 nm. Lower panel: coomassie blue staining of the peak fractions shown on the top following SDS-PAGE. M, molecular weight ladder (kDa). Numbers on top of SDS-PAGE panel indicate elution volumes. c Measurement of the binding affinity between AtPRK6^LRR and AtLURE1.2 by ITC. Left upper panel: twenty injections of AtLURE1.2 solution were titrated into AtPRK6^LRR solution in the ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Left lower panel: the binding isotherm for AtPRK6^LRR-AtLURE1.2 interaction. The integrated heat is plotted against the molar ratio between AtLURE1.2 and AtPRK6^LRR. Data fitting revealed a binding affinity of about 3.1 μM. Right panel: binding affinity between AtPRK3^LRR and AtLURE1.2 by ITC

Fig. 2

Overall structure of the AtPRK6^LRR-AtLURE1.2 complex. a Structure of the AtPRK6^LRR-AtLURE1.2 complex shown in different modes. Left: both AtPRK6^LRR (pink) and AtLURE1.2 (cyan) are shown in cartoon. The disulfide bond Cys229−Cys237 is shown in yellow and three residues capping the hydrophobic core of the last LRR of AtPRK6^LRR are shown in stick and labeled. ‘N’ and ‘C’ represent N- and C-terminus, respectively. Middle: the C-terminal loop of AtPRK6^LRR binds a positively charged surface of AtLURE1.2. AtLURE1.2 is shown in electrostatic surface. Red, blue, and white represent negative, positive, and neutral surfaces, respectively. Right: interaction between AtLURE1.2 and AtPRK6^LRR is both shape-complementary and charge-complementary. Both AtLURE1.2 and AtPRK6^LRR are shown in electrostatic surface. b Sequence alignment of AtPRK6^LRR, AlPRK6^LRR, CrPRK6^LRR, and AtPRK3^LRR. Conserved and similar residues are boxed with red ground and red font, respectively. Two cysteine residues forming a disulfide bond are indicated by the same green numbers. The AtLURE1.2-interacting amino acids of AtPRK6^LRR are highlighted with blue squares on top

Fig. 3

Recognition mechanism of AtLURE1.2 by AtPRK6^LRR. a A close-up view of binding of the C-terminal side from AtPRK6^LRR to a positively charged surface of AtLURE1.2. AtLURE1.2 is shown in transparent electrostatic surface. Some of the critical amino acids from AtPRK6^LRR and AtLURE1.2 are shown in stick. Squares in three different colors indicate the interfaces between AtPRK6^LRR and AtLURE1.2. b The C-terminal side of AtPRK6^LRR binds to a hydrophobic cavity formed between the α-helix and the second β-sheet of AtLURE1.2. Red lines indicate hydrogen bonds. The red sphere represents water molecule. c Arg83 of AtLURE1.2 is at the center of the AtLURE1.2-AtPRK6^LRR interface and form extensive interactions with AtPRK6^LRR. d A network of polar interactions is formed between Asp234 of AtPRK6 and AtLURE1.2

Fig. 4

Mutagenesis analysis and pollen tube attraction with mutant AtLURE1.2 and AtPRK6. a Effect of AtLURE1.2 mutations on the interaction with AtPRK6^LRR. The assay was performed as described in Fig. 1a. b Effect of AtPRK6^LRR mutations on the interaction with AtLURE1.2. The assay was performed as described in Fig. 1a. c Attraction frequencies of pollen tubes by mutant AtLURE1 peptides. Attraction frequencies of semi-in-vivo pollen tubes were examined using gelatin beads containing 250 nM AtLURE1.2 peptides of wild-type and mutants, respectively. For each assay, 9–16 pollen tubes were examined, and assays were repeated 3−5 times in each condition. Data are mean and s.d., and numbers of assays (replicates) in each condition were indicated. An asterisk indicates statistical significance among wild-type and mutant AtLURE1.2 peptides (Tukey−Kramer test; P < 0.05). The frequency in R83A AtLURE1.2 was significantly lower than those in other four peptides. d Pollen tubes attraction in gelatin-beads assay. Photographs just after putting beads (0 min) and after attraction are shown. Arrowheads indicate the tip position when a bead was placed and arrows indicate the tip of attracted pollen tubes at the indicated time. The data are representative of 20−25 images. In the assay using R83A AtLURE1.2, more than 44% pollen tubes were not attracted to the bead. Scale bars are 20 μm. e Attraction frequencies of pollen tubes with mutations in PRK6 receptor. Attraction frequencies of semi-in-vivo pollen tubes (prk6 transformed with PRK6 of wild-type, D234A, N239A, and ΔN239I240) were examined using gelatin beads containing 250 nM wild-type AtLURE1.2 peptides. For each assay, 10–23 pollen tubes were examined, and repeated for three times in each condition. Frequencies in D234A and ΔN239I240 PRK6 pollen tubes were significantly lower than those in wild-type and N239A PRK6 pollen tubes. f Pollen tubes attraction in gelatin-beads assay. Photographs just after putting beads (0 min) and after attraction are shown. Arrowheads indicate the tip position when a bead was placed and arrows indicate the tip of attracted pollen tubes at the indicated time. The data are representative of 11−14 images. Scale bars are 20 μm