Structural basis of a light-activated signaling pathway involved in Brucella virulence

JIMENA RINALDI; MARIANA GALLO; DANIEL O. CICERO; SEBASTIÁN KLINKE; GASTÓN PARIS; FERNANDO A. GOLDBAUM

Buenos Aires

Congreso; Brucellosis 2011International Research Conference; 2011

Asociación Argentina de Microbiología

Light affects the virulence  of  Brucella abortus, the etiologic  agent of brucellosis. The photoreceptor that mediates this effect is the  protein LOV-HK. This protein contains an N-terminal photosensory LOV domain, a  PAS domain and a C-terminal histidine kinase domain. The molecular mechanism of  the light sensing in bacteria is unknown. LOV domains consist of an alfa-helix,  called J-helix, which is involved in the transduction of the signal to the  output domain of the molecule. Light absorption causes formation of a covalent  bond between a conserved Cys residue and the C(4a) atom from the FMN ring.  While the covalent bond formation is fast (in µs) in all LOV proteins  characterized, the recovery to the dark state varies among different  LOV-containing proteins, ranging from 20-30 seconds in phototropins to hours in  some prokaryotic photoreceptors. In Brucella LOV-HK the adduct state is  extremely stable (does not decay significantly even for days). However,  absorption spectra and 1D-H NMR studies show that a Brucella LOV  domain fragment comprising only the core completes the photocycle  after several hours. The addition of only 20 residues corresponding to the  J-helix to this core abrogates the dark recovery, showing the same truncated  photocycle as the full-length LOV-HK protein. These findings indicate that the  J-helix is not just a transducer of the signal to the HK domain but also an  element of the sensor domain that determines its photochemical behavior. The  crystal structure of the Brucella LOV  domain shows the canonical fold previously observed for other LOV domains,  comprising a five-stranded antiparallel β-sheet and four alfa-helices.  The molecule was trapped in the dark state. The residues that interact with FMN  are conserved. The N-terminal part of the J-helix, included in the crystal  structure, extends apart from the core and into the solvent. NMR dynamics  relaxation measurements show that the domain is rigid under dark conditions,  however conformational instability was detected in the interface between  monomers, shown in the crystal structure. There are not big conformational  changes upon illumination, but several residues suffer changes in their  chemical shift. Most of them are contacting FMN and others are not, among which  there must be the key elements in the signal transduction to the effector  domain.