The Extracellular Domain of Pollen Receptor Kinase 3 is structurally similar to the SERK family of co-receptors

Abstract

During reproduction in flowering plants, the male gametophyte delivers an immotile male gamete to the female gametophyte in the pistil by formation of pollen tubes. In Arabidopsis thaliana, two synergid cells situated on either side of the egg cell produce cysteine-rich chemoattractant peptide LURE that guides the pollen tube to the female gametophyte for sexual reproduction. Recently, in Arabidopsis thaliana, Pollen Receptor Kinase 3 (PRK3), along with PRK1, PRK6, and PRK8, have been predicted to be the receptors responsible for sensing LURE. These receptors belong to the Leucine Rich Repeat Receptor Like Kinases (LRR-RLKs), the largest family of receptor kinases found in Arabidopsis thaliana. How PRKs regulate the growth and development of the pollen tube remains elusive. In order to better understand the PRK-mediated signaling mechanism in pollen tube growth and guidance, we have determined the crystal structure of the extracellular domain (ecd) of PRK3 at 2.5 Å, which resembles the SERK family of plant co-receptors. The structure of ecdPRK3 is composed of a conserved surface that coincides with the conserved receptor-binding surface of the SERK family of co-receptors. Our structural analyses of PRK3 have provided a template for future functional studies of the PRK family of LRR-RLK receptors in the regulation of pollen tube development.

Figure 1

Structure of the extracellular domain of Arabidopsis thaliana PRK3. (A) PRK3 contains an N-terminal signal peptide domain (SP, residues 1–19), an LRR capping domain (CD, 20–54), a leucine rich repeat domain (LRR, 65–209) which harbors 6 plant-specific LRRs, an LRR C-terminal domain (CT, 210–249), a transmembrane domain (TM, 250–270), and an intracellular kinase domain (KD, 358–633). (B) The structure of PRK3 (residues 20–237) is represented as a cartoon representation. The CD is colored in red, LRR domain in cyan, and CT in blue. Each LRR is numbered in the N-terminal β-strand of the repeat. Two glycosylated asparagine residues and the four-cysteine residues that form two disulfide bonds of PRK3 are depicted as stick representations and the disulfide bonds are colored in yellow. (C) Sequence alignment of the six LRRs in the ectodomain of PRK3; the conserved residues are colored in red. The conserved LRR motif is shown on the top of the alignment where “X” stands for any residue. (D) Surface representation of the PRK3 structure where the positively charged surfaces are depicted in blue, and negatively charged surfaces are colored in red. (E) The asymmetric unit of the PRK3 crystal contains two chains of the PRK3 molecule, with the A chain colored in cyan and B chain shown in red.

Figure 2

Two crystal packing dimers of the PRK3 structure. (A) A crystallographic packing dimer between the same chains (chain A) is depicted in cyan as a cartoon representation, and the residues facilitating the packing interactions are shown in either red or blue with side chains depicted as sticks. (B) Cartoon representation of the crystallographic packing dimer between the two different chains (chain A and B), where chain A is depicted in cyan and chain B is in red. The residues mediating the packing interactions are shown in either red or blue with the side chains depicted as sticks. (C) Size-exclusion chromatogram of the expressed ecdPRK3 recombinant protein. The retention volumes of proteins used as molecular weight standards are indicated on top.

Figure 3

Structural alignment between AtPRK3 and the SERK family of plant co-receptor structures. (A) Structure of AtPRK3 is superimposed on BAK1 (PDB ID: 4M7E) with a root-mean-square deviation (RMSD) of 1.92 Å in 154 over 208 residues. The PRK3 structure is colored in cyan and the BAK1 structure is in red. (B) Structure of PRK3 is superimposed on SERK1 (PDB ID: 4LSX) with an RMSD of 1.89 Å in 157 over 208 residues. The PRK3 structure is colored in cyan and the SERK1 structure is in magenta. (C) Superposition of the ectodomain structures of PRK3 and SERK2 (PDB ID: 5GQR) with an RMSD of 1.96 Å in 159 over 208 residues. The PRK3 structure is colored in cyan and the SERK2 structure is in orange. (D) Amino acid sequence of the Arabidopsis thaliana PRK3 extracellular domain is aligned with the SERK family of Arabidopsis thaliana co-receptors, BAK1, SERK1, and SERK2. Residue numbers of A. thaliana PRK3 are indicated on the top of the sequences. The residues that are identical in all four sequences are colored in red. The residues that are similar in all four sequences are colored in blue. Similar residues are defined as: (1) negatively charged side chains as D and E; (2) positively charged side chains as R and K; (3) aliphatic side chains as L, I, and V; (4) aromatic side chains as F, Y, and W; (5) side chains with hydroxyl group as S and T; (6) amide side chains as Q and N. The definition of similar residues is adapted from the BLOSUM matrix. The sequence identity between ecdPRK3 and ecdBAK1, ecdSERK1, and ecdSERK2 are 32.43%, 32.32%, and 30.81% respectively.

Figure 4

AtPRK3 contains a conserved surface patch that resembles the conserved receptor-binding surface of the SERK family of plant co-receptors. (A) The conserved residues that are either identical or similar in all seven selected PRK3 orthologues (in panel E) are colored in red on the molecular surface of PRK3 structure, which is depicted in cyan. (B) The interface residues of BAK1 that mediate its interaction with BRI1 (pdb id: 4m7e) are colored in blue on the molecular surface of BAK1, which is shown in red. (C) The interface residues of SERK1 that mediate its interaction with BRI1 (pdb id: 4lsx) are colored in blue on the molecular surface of SERK1, which is shown in magenta. (D) The interface residues of SERK2 that mediate its interaction with PXY (pdb id: 5gqr) are colored in blue on the molecular surface of SERK2, which is shown in orange. (E) The amino acid sequences of the extracellular domains of the seven selected PRK3 orthologs are aligned. at, cs, rs, br, al, hu, cc, stand for Arabidopsis thaliana, Camelina sativa, Raphanus sativus, Brassica rapa, Arabidopsis lyrata, Herrania umbratica, and Cajanus cajan, respectively. The overall sequence identity between the extracellular domain of Arabidopsis thaliana PRK3 and that of the PRK3 of Camelina sativa, Raphanus sativus, Brassica rapa, Arabidopsis lyrata, Herrania umbratica, and Cajanus cajan is 85%, 81%, 77%, 68%, 48%, and 41%, respectively. The residue numbers of A. thaliana PRK3 are indicated on the top the sequences. The residues that are identical in all seven orthologues are colored in blue. The residues that are similar in all seven sequences are colored in cyan. Similar residues are defined as: (1) negatively charged side chains as D and E; (2) positively charged side chains as R and K; (3) aliphatic side chains as L, I, and V; (4) aromatic side chains as F, Y, and W; (5) side chains with hydroxyl group as S and T; (6) amide side chains as Q and N. The definition of similar residues is adapted from the BLOSUM matrix.

Figure 5

Sequence alignment of the extracellular domains of Arabidopsis thaliana PRK1-8. The amino acid sequences of the extracellular domains of A. thaliana PRK1-8 are aligned. Each LRR repeat of PRK3 is indicated at the bottom of the sequences. The secondary structural elements of PRK3 are shown on the top with α as α-helix, β as β-strand, η as 3₁₀ helix, and TT and turn. The conserved residues are colored in red. The two pairs of cysteine residues that form disulfide bonds in PRK3 structure are indicated in green numerical below the sequence. The AtLURE1.2 interacting residues of PRK6 are indicated with the black solid triangles underneath the sequence.