Receptor Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Signaling via Protein‐Protein Cross Talk

Abstract

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+by increasing concentration of acP yielded lower KM [~21mM] compared to PhoB [~8mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking and GST-assay using various length of ASR [full length, 1–261, and truncated (1–229, 1–239, 1–249)]. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. Recent results of STK 3169 cloning, optimized expression, purification and subsequent characterization in phosphorylation assay will be presented. Support or Funding Information Supported by NIH-NIGMS Support for Competitive Research, SCORE SC3 award GM113803 to VT This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.