Mutational approches to obtain locked conformations of the bacteriophytochrome from the plant pathogen Xanthomonas campestris

ANTELO, GIULIANO; GOLDBAUM, FERNANDO A.; RINALDI, JIMENA; SANCHEZ, MAXIMILIANO; BONOMI, HERNAN R.; KLINKE, SEBASTIÁN; OTERO, LISANDRO HORACIO

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

SAIB, SAIC, SAI, SAA, SAB, SAB, SAFE, SAFIS, SAH, SAP

Light modulates the host-microbe interaction and several virulence-related phenotypes in the plant pathogen Xantomonas campestris. This is mediated by its bacteriophytochrome, XccBphP.Bacteriophytochromes are photoreceptors found in bacteria that bind biliverdin (BV) as their chromophore. They typically present a red-absorbing form (Pr) and a far-red absorbing form (Pfr). XccBphP is a bathy-like phytochrome. This means that it exhibits a dark conversion from Pr to Pfr, reaching an equilibrium Pfr:Pr ratio of 85:15 (pH 7.7) with a half-life of 7.45 h.XccBphP presents at the N-terminus a PAS2-GAF-PHY domain triad as its photosensory module, and a PAS9 domain as its output module. In order to get insight into the light-dependent activation mechanisms, the crystal structure of the full version of XccBphP was recently solved by our group. The solved structure shows a parallel homodimer arrangement along a central helical spine in the Pr state. With the aim of obtaining XccBphP versions locked in Pfr or Pr states to perform molecular, structural and physiological studies, we are carrying out rational and random mutagenesis approaches in several key positions likely to affect its photochemical behavior. The analysis is accomplished by UV-VIS spectroscopy.By this means, we have identified the mutant G454E, which exhibits a 100% Pfr conversion in the dark. This is reminiscent of the monomeric ΔPAS9 mutation. Hence, we performed a static light scattering determination of its molecular weight. The results show that the oligomeric state of the mutant is a dimer. Thus, we have obtained a full-length dimeric variant with a stable (but not locked) Pfr state. Currently, a crystallogenesis screening of XccBphP G454E in dark conditions is being performed. We hope to obtain a crystal structure in the Pfr state to compare it to the already solved structure and elucidate the light-driven conformational changes.