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Homology between DUF784, DUF1278 domains and the plant prolamin superfamily typifies evolutionary changes of disulfide bonding patterns

protein families to generate structural and functional novelty. The DUF784 and DUF1278 domains were initially identified by automatic processing by the Pfam database. Recently, using genome-wide expression profiling, Jones-Rhoades et al. have shown that many members of these two domain families are functionally involved in plant embryo sac development or fertilization. Firstly, they are downregulated in ovules of determinate infertile 1 (dif1) mutants, which represent a genetic ablation of the entire embryo sac. Secondly, DUF784 and DUF1278 genes are found to be expressed in synergid cells and also subjected to the regulation of the Myb98 transcription factor. In the ovules of myb98 mutants, many DUF784 genes are downregulated whereas many DUF1278 genes are upregulated. However, since both DUF784 and DUF1278 domains lack homology, biochemical and structural information, their specific function is still unclear. In the current study, as an effort to define the putative function for the uncharacterized domain families (for example, domains of unknown function, DUFs) in the Pfam database, the evolutionary and functional predictions about DUF784 and DUF1278 domains are presented. For these two domains, the sensitive PSI-BLAST program could not retrieve any sequence other than their own members. We then used fold recognition strategy via meta server (http://meta.bioinfo.pl/) to identify their remotely related sequences. Meta server assembles various state-of-the-art fold recognition programs including FUGUE and FFAS03, and has been approved powerful in protein fold assignment. An Arabidopsis thaliana (At) DUF784 domain (GI: 145332721) was firstly used as a query in the meta server, which returned several structural hits identified by FFAS03 and FUGUE programs with the consensus scores between 37 and 41. Domain or structural fold annotation for the retrieved hits revealed that they all belong to the prolamin superfamily. Similar structural hits belonging to the prolamin superfamily were also retrieved for the DUF1278 domain by these programs. Although these 3D-Jury scores are below the cutoff of 50 (corresponding to confident prediction), previous studies have shown that relatively low scores between 30 and 45 still could successfully assign remote homology in some cases. Multiple sequence alignment (upper row in Fig. 1) has shown that all the members of the prolamin superfamily contain eight conserved Cys residues which are spaced as follows C 1 ...C 2 ...C 3 C 4 ...C 5 XC 6 ...C 7 ... C 8 (C, Cys). These Cys residues can form four pairs of disulfide bonds: C 1 -C 5 , C 2 -C 3 , C 4 -C 7 , C 6 -C 8 , which have shown to be critical for maintenance of tertiary structure folded by four central helices. In contrast, both DUF784 and DUF1278 domains are characterized by the six conserved Cys residues. Although there is very low sequence similarity between DUF784, DUF1278 and the prolamin superfamily, sequence profile alignment guided by secondary structure information has assigned the conserved Cys residues of both DUF784 The prolamin superfamily (also called protease inhibitor/lipid-transfer protein/ seed storage 2S albumin family; Pfam ID: PF00234) represents one of the most wide-spread protein superfamilies in plant genome and comprises 2S albumin storage proteins, non-specific lipid transfer proteins (nsLTPs), alpha-amylas/trypsin inhibitors (AAI/ATI), prolamins, indulines, grain softness proteins, Hageman factor inhibitor (HFI), and Arachis hypogaea 6 (Arah6) allergen. The members of the prolamin superfamily are involved in seed storage (2S albumin and prolamins), natural defenses of plants against insects and pathogens such as fungi and bacteria (nsLTPs, AAI/ATI), and also constitute major source of food allergens to humans (AAI/ATI, nsLTPs, Arah6). In the present study, by using sequence analysis and fold recognition methods, functionallyunknown families DUF784 (Pfam ID: PF05617) and DUF1278 (Pfam ID: PF06915) were classified into two unique versions of the prolamin superfamily. In contrast, to most members of the prolamin superfamily that contain eight conserved cysteine (Cys) residues forming four pairs of disulfide bond, these two new domains only contain six Cys resides whose counterparts in canonical prolamin members can form three typical pairs of disulfide bonds. Thus the gain or loss of Cys-Cys paris and the associated changes of disulfide bonding patterns among and between the prolamin superfamily, DUF784 and DUF1278 domains represent a typical evolutionary manner for