Structure of SPH (self-incompatibility protein homologue) proteins: a widespread family of small, highly stable, secreted proteins.

SPH (self-incompatibility protein homologue) proteins are a large family of small, disulfide-bonded, secreted proteins, initially found in the self-incompatibility response in the field poppy (Papaver rhoeas), but now known to be widely distributed in plants, many containing multiple members of this protein family. Using the Origami strain of Escherichia coli, we expressed one member of this family, SPH15 from Arabidopsis thaliana, as a folded thioredoxin fusion protein and purified it from the cytosol. The fusion protein was cleaved and characterised by analytical ultracentrifugation, circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. This showed that SPH15 is monomeric and temperature stable, with a ß-sandwich structure. The four strands in each sheet have the same topology as the unrelated proteins: human transthyretin, bacterial TssJ and pneumolysin, with no discernible sequence similarity. The NMR-derived structure was compared with a de novo model, made using a new deep learning algorithm based on co-evolution/correlated mutations, DeepCDPred, validating the method. The DeepCDPred de novo method and homology modelling to SPH15 were then both used to derive models of the 3D structure of the three known PrsS proteins from P. rhoeas, which have only 15-18% sequence homology to SPH15. The DeepCDPred method gave models with lower discreet optimised protein energy scores than the homology models. Three loops at one end of the poppy structures are postulated to interact with their respective pollen receptors to instigate programmed cell death in pollen tubes.

1H, 13C and 15N NMR assignments of self-incompatibility protein homologue 15 from Arabidopsis thaliana.

The SPH proteins are a large family of small, disulphide-bonded, secreted proteins, originally found to be involved in the self-incompatibility response in the field poppy (Papaver rhoeas). They are now known to be widely distributed in plants, many containing multiple members of this protein family. Apart from the PrsS proteins in Papaver the function of these proteins is unknown but they are thought to be involved in plant development and cell signalling. There has been no structural study of SPH proteins to date. Using the Origami strain of E. coli, we cloned and expressed one member of this family, SPH15 from Arabidopsis thaliana, as a folded thioredoxin-fusion protein, purified it from the cytosol, and cleaved it to obtain the secreted protein. We here report the assignment of the NMR spectra of SPH15, which contains 112 residues plus three N-terminal amino acids from the vector. The secondary structure propensity from TALOS+ shows that it contains eight beta strands and connecting loops. This is largely in agreement with predictions from the amino acid sequence, which show an additional C-terminal strand.