Crystal structure of a putative phosphate binding protein from Synechocystis sp. PCC 6803 reveals an evolutionary hotspot.

Phosphorus is an essential element for all living organisms and is often a limiting nutrient in natural environments. Phosphate-binding proteins (PBPs) are defined by their ability to bind phosphorus-containing substrates with high affinity, facilitating the uptake and transport of this critical element across cellular membranes; thus, they play a vital role in phosphorus acquisition from external environments. In this study, we report the crystal structure of a PBP encoded by the gene sll0540 from Synechocystis sp. PCC 6803, in complex with phosphate. The protein exhibits a structure typical of ABC phosphate transport receptors, with the phosphate fully contained within a cleft formed by two domains, similar to other PBPs. However, biochemical assays indicate that Sll0540 possesses a relatively low phosphate-binding affinity (K = 223.7 μM) compared to other PBPs. Structural comparisons reveal that Asp225, a proton acceptor positioned differently than in other analogs, forms a short hydrogen bond directly coordinating with the bound phosphate. Furthermore, site-directed mutagenesis showed that mutating Asp225 to Asn or Ser significantly improves the phosphate binding affinity of Sll0540 to varying degrees. Our findings identify an evolutionary hotspot in Synechocystis sp. PCC 6803 that may contribute to the development of PBPs with enhanced phosphate binding affinities.