Structural modules for receptor dimerization in the S -locus receptor kinase extracellular domain

Abstract

The highly polymorphic S-locus receptor kinase (SRK) is the stigma determinant of specificity in the self-incompatibility response of the Brassicaceae. SRK spans the plasma membrane of stigma epidermal cells, and it is activated in an allele-specific manner on binding of its extracellular region (eSRK) to its cognate pollen coat-localized S-locus cysteine-rich (SCR) ligand. SRK, like several other receptor kinases, forms dimers in the absence of ligand. To identify domains in SRK that mediate ligand-independent dimerization, we assayed eSRK for self-interaction in yeast. We show that SRK dimerization is mediated by two regions in eSRK, primarily by a C-terminal region inferred by homology modeling/fold recognition techniques to assume a PAN APPLE-like structure, and secondarily by a region containing a signature sequence of the S-domain gene family, which might assume an EGF-like structure. We also show that eSRK exhibits a marked preference for homodimerization over heterodimerization with other eSRK variants and that this preference is mediated by a small, highly variable region within the PAN APPLE domain. Thus, the extensive polymorphism exhibited by the eSRK not only determines differential affinity toward the SCR ligand, as has been assumed thus far, but also underlies a previously unrecognized allelic specificity in SRK dimerization. We propose that preference for SRK homodimerization explains the codominance exhibited by a majority of SRKs in the typically heterozygous stigmas of self-incompatible plants, whereas an increased propensity for heterodimerization combined with reduced affinity of heterodimers for cognate SCRs might underlie the dominant-recessive or mutual weakening relationships exhibited by some SRK allelic pairs.self-incompatibility ͉ ligand-independent receptor dimerization ͉ yeast two-hybrid T he S-locus receptor kinase (SRK) is a single-pass integral plasma membrane protein of the stigma epidermis that functions in the recognition and rejection of self-related pollen in self-incompatible members of the Brassicaceae (reviewed in refs. 1 and 2). The SRK receptor and its pollen-coat localized ligand, the S-locus cysteine-rich protein (SCR) (1), also designated SP-11 (2), are encoded by tightly linked and highly polymorphic genes within the S-locus haplotype. Specificity in the self-incompatibility response derives from allele-specific SRK-SCR interaction (3, 4). On self-pollination, the binding of SCR to the extracellular domain of its cognate SRK activates the receptor and triggers an intracellular signaling cascade that leads to arrest of ''self'' pollen tubes. In contrast, pollen tube growth in cross-pollinations proceeds unimpeded because ''nonself'' SCR cannot bind SRK. In view of this high degree of specificity in the affinity of SRK for its SCR ligand, the extensive polymorphisms exhibited by the SRK extracellular region (eSRK) have heretofore been considered in the context of specificity in the SRK-SCR interaction.The eSRK does more than interact with SCR, however. SRK forms oligo...

Structural basis for specific self-incompatibility response in Brassica

“…The configuration of the β-strands in both of these domains has an approximate three-fold internal symmetry, forming a "Y" or trefoil-shaped structure. As previously predicted [17], a database search using the DALI server revealed that these two lectin domains share appreciable structural similarity to a mannose-recognizing lectin from Japanese Marasmius oreades [24] despite their weak sequence similarity to the latter lectin ( Figure 2B and Supplementary information, Figure S1A). However, the hairpin loops of these proteins vary greatly in length, sequence, and conformation ( Figure 2B and Supplementary information, Figure S1A).…”

“…A modeling study had suggested that residues 293-346 of eSRK6 encode an EGF-like domain [17] that is principally defined by six cysteines, which are known to form disulphide bonds in a 1-3, 2-4, and 5-6 pattern [25]. Indeed, this region of eSRK9 forms a structure that can be largely aligned with that of EGF [26] ( Figure 2C).…”

“…SRK is the prototypic member of the S-domain RLK (SD-RLK) subfamily with ~40 encoded in the Arabidopsis thaliana genome [16]. The eSRK is characterized by two contiguous N-terminal lectin-like domains followed by a region containing 12 conserved cysteine residues [17]. Structural modeling predicted the six N-terminal cysteines to be contained within an EGF-like domain and the six C-terminal cysteines within a PAN/APPLE domain [17].…”

“…The eSRK is characterized by two contiguous N-terminal lectin-like domains followed by a region containing 12 conserved cysteine residues [17]. Structural modeling predicted the six N-terminal cysteines to be contained within an EGF-like domain and the six C-terminal cysteines within a PAN/APPLE domain [17]. Comparison of eSRK sequences identified several hyper-variable (hv) regions, designated hvI, hvII and hvIII, which were predicted to be important for SI specificity [18,19].…”

Self-Incompatibility in the Brassicaceae

“…Interestingly, an artifi cially expressed dimerized form of eSRK exhibited high-affi nity binding to SP11. Another recent study suggested that two regions in the extracellular domain of SRK mediated the homo-dimerization of eSRK (Naithani et al 2007 ). Taken together, these studies suggested that SRK on the stigmatic membrane is in an equilibrium between the inactive monomeric or dimeric low-affi nity forms and the dimeric active high-affi nity form, and that the SP11/SCR binding to its cognate SRK stabilizes its dimeric active form, which is expected to trigger the SI responses in the papilla cell (Shimosato et al 2009 ) (Fig.…”