In Silico Analysis of Putative Paralytic Shellfish Poisoning Toxins Export Proteins in Cyanobacteria

Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na+ channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H+) or sodium (Na+) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na+ and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na+ recognition domain was conserved in both SxtF/M, indicating that Na+ can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na+ incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na+ in toxin export, as well as the motifs L398XGLQD403 (SxtM) and L390VGLRD395 (SxtF) in toxin recognition.麻痹性贝类毒素（Paralytic shellfish poisoning toxins, PSTs）是一类由甲藻和蓝细菌合成的超过30种天然生物碱家族，其对动物的毒性通过阻断电压门控钠离子通道（voltage-gated Na+ channel）介导。PST类似物的外排可能由SxtF和SxtM介导，这两种为推定的多药与有毒化合物外排（multidrug and toxic compound extrusion, MATE）家族转运蛋白，编码于PSTs生物合成基因簇（biosynthetic gene cluster, sxt）中。sxtM存在于所有已分析的sxt基因簇中；然而sxtF仅存在于柱胞藻-尖头藻进化支（Cylindrospermopsis-Raphidiopsis clade）中。这类转运蛋白的能量供应依赖于跨膜质子（H+）或钠离子（Na+）的电化学梯度。由于PSTs的具体功能仍未明确，且产PSTs生物体的遗传操作方法尚未建立，蛋白质结构分析将有助于解析其功能。通过分析8株产PSTs蓝细菌的sxt基因簇，我们未发现sxtF或sxtM的存在与特定PSTs谱型之间存在相关性。对SxtF/M的系统发育分析显示，SxtF在柱胞藻-尖头藻进化支中具有高度保守性，提示其底物亲和性较为保守。副溶血性弧菌（Vibrio parahaemolyticus）与霍乱弧菌（V. cholerae）的NorM蛋白中，参与钠离子与药物识别的两个结构域，在SxtF/M中同样存在。钠离子识别结构域在SxtF和SxtM中均保守，表明钠离子可维持其作为阳离子反向转运体（cation anti-transporter）的功能。毒素结合的共有基序在SxtF与SxtM之间存在差异，提示二者的底物结合特性存在差异。通过蛋白质建模与分子对接分析，我们发现识别结构域与特定PST类似物之间并无显著亲和力。这与我们此前在布鲁氏尖头藻（R. brookii）D9菌株中开展的PST外排研究结果一致，当时我们观察到钠离子孵育对不同PST类似物的响应较为相似。上述结果进一步证实了"钠离子参与毒素外排"的假说，以及L398XGLQD403（SxtM）和L390VGLRD395（SxtF）基序在毒素识别中的作用。