Mutational approaches to obtain locked conformations of the bacteriophytochrome from the plant pathogen Xanthomonas campestris (Póster)

GIULIANO T. ANTELO; SEBASTIÁN KLINKE; MAXIMILIANO SÁNCHEZ-LAMAS; FERNANDO A. GOLDBAUM; LISANDRO OTERO; HERNÁN R. BONOMI; JIMENA RINALDI

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017

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

Lightmodulates the host-microbe interaction and several virulence- relatedphenotypes in the plant pathogen Xantomonas campestris. This is mediated by itsbacteriophytochrome, XccBphP. Bacteriophytochromes are photoreceptors found inbacteria that bind biliverdin (BV) as their chromophore. They typically presenta red-absorbing form (Pr) and a far-red absorbing form (Pfr). XccBphP is abathy-like phytochrome. This means that it exhibits a dark conversion from Prto Pfr, reaching an equilibrium Pfr:Pr ratio of 85:15 (pH 7.7) with a half-lifeof 7.45 h. XccBphP presents at the N-terminus a PAS2-GAF-PHY domain triad as itsphotosensory module, and a PAS9 domain as its output module. In order to getinsight into the light-dependent activation mechanisms, the crystal structureof the full version of XccBphP was recently solved by our group. The solvedstructure shows a parallel homodimer arrangement along a central helical spinein the Pr state. With the aim of obtaining XccBphP versions locked in Pfr or Prstates to perform molecular, structural and physiological studies, we arecarrying out rational and random mutagenesis approaches in several keypositions likely to affect its photochemical behavior. The analysis isaccomplished by UV-VIS spectroscopy. By this means, we have identified themutant G454E, which exhibits a 100% Pfr conversion in the dark. This isreminiscent of the monomeric ΔPAS9 mutation. Hence, we performed a static lightscattering determination of its molecular weight. The results show that the oligomericstate of the mutant is a dimer. Thus, we have obtained a full-length dimericvariant with a stable (but not locked) Pfr state. Currently, a crystallogenesisscreening of XccBphP G454E in dark conditions is being performed. We hope toobtain a crystal structure in the Pfr state to compare it to the already solvedstructure and elucidate the light-driven conformational changes.