Light-induced subunit dissociation by a light-oxygen-voltage domain photoreceptor from Rhodobacter sphaeroides

Abstract

Light-oxygen-voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of anti-sigma factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV domain protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N- and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light- and dark-state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin-cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single-residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb the oligomeric state. Size exclusion chromatography, multiangle light scattering, small-angle X-ray scattering, and cross-linking studies indicate that RsLOV dimerizes in the dark but, upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove to be useful for engineering molecular associations in controlled cellular settings.

Figure 1

LOV domain homology and chemistry (A) Structure-based sequence alignment of LOV domains, Avena sativa LOV2 (2V1A), Bacillus subtilis (2PR5), Chlamydomonas reinhardtii (1N9L), Pseudomonas putida (3SW1), Rhodobacter sphaeroides, Neurospora crassa Vivid. (2PD7), and Erythrobacter litoralis (3P7N). Secondary structure elements noted above (α-helical residues in blue, β-sheet in aqua). Residues critical to flavin coordination are highlighted in yellow, sites of RLOV single residue variants are highlighted in pink, and the four Ala repeat in Jα and Kα helices are highlighted in green. (B) Domain arrangement of sequence aligned LOV domain proteins (number of residues specified). HisK, histidine kinase; Ser/ThrK, serine/threonine kinase; STAS, sulfate transporter anti-s antagonist; HTH, helix-turn-helix. (C) Schematic of light-induced cysteinyl-FMN (C4a) covalent adduct formation. (D) Genetic context of LOV domain protein in R. sphaeroides.

Figure 2

LOV subunit structures. (A) RsLOV structure in the dark state, with PAS core in violet, N-terminal extension in orange, and C-terminal extension in light green. Dark state structures of N. crassa VVD (2PD7, PAS core light orange), A.sativa LOV2 (2V1A, PAS core forest green), C. reinhardtii LOV1 (1N9L, PAS core light blue) B. subtilis LOV (2PR5, PAS core pink), EL222 (3P7N, PAS core aqua, HTH motif gray), P. putida (3SW1, PAS core salmon) with N-terminal extensions in orange, and C-terminal extensions in light green.

Figure 3

Comparison of LOV dimers: Light state structures of N. crassa VVD (3RH8, light orange), dark state structure of B. subtilis LOV (2PR5, pink), light state structure of P. putida (3SW1, salmon), and dark state structure of RsLOV (purple), N-terminal extensions in orange, C-terminal extensions in light green.

Figure 4

The RsLOV dimerization motif. (A) Four helix bundle at interface of the RsLOV crystallographic dimer, A and B subunits distinguished by color. (B) Side chain contacts at Jα interface of chain A and chain B (C) Side chain contacts at the Jα and Kα interface. There are salt bridge between Arg145 and Glu142 (same monomer) and between Arg15 and Glu154 (opposing monomer). Hydrogen bonding interactions range from 2.45 to 3.66 Å and also include the amide H of Arg158 to the backbone O of Gly17 (opposing monomer), and the amide H of Arg159 to the side chain O of Ser124 (opposing monomer). The residues with the most buried surface area are within the crossing point of the two Jα helices at the region of the four alanine repeat: Ala130–133, Gly134, Gly137, Ala138, and the cross-section of Kα including Glu154, Arg158, and Ala162.

Figure 5

Solution properties of RsLOV. (A) Elution profile of RsLOV expressed in E. coli with a Supredex 75 10/300 column. Light state (red, dashed) elutes at 16.1 mL, dark state (black, solid) elutes at 15.5 mL. (B) Electronic absorption spectra of light state RsLOV (red, dashed), and dark state RsLOV (black, solid).

Figure 6

Irridiation of RsLOV crystals. (A) Light state structure of RsLOV with electron density corresponding to the light-induced cysteinyl-C4a adduct of Cys55 and FMN (F_o-F_c simulated annealing omit map, obtained by omitting the flavin molecule and Cys55 residue, contoured at 2.2σ in cyan, 0.7σ in gray). The two conformations of Cys55B and flavin are shown in yellow (covalently bound) and green (unbound). (B) FMN binding pocket residues affected by changes in light state conversion. (C) RsLOV structure in the light state with F_o–F_o difference map of light-dark amplitudes (green is +3.5σ, red is −3.5σ).

Figure 7

RsLOV point mutants. (A) Dark state structure of RsLOV with residues adjacent to the FMN binding pocket targeted for point mutations highlighted in cyan. (B) Residues targeted for point mutations at the RsLOV dimer interface highlighted: S127 (yellow), E129 (blue), A136 (green), A138 (aqua), E142 (pink), R145 (orange), A167 (red).

Figure 8

Structures of L32V and A138Y RsLOV. (A) Structure of L32V RsLOV variant in the dark state colored by B factor (ROYGBIV spectrum coloring in which red is the maximum and violet is the minimum value) overlayed on the P6₅ dark state RLOV structure (gray, transparent). (B) Structure of the A138Y RsLOV variant in the dark state (aqua) overlaid on the P6₅ dark state RsLOV structure (violet). Dimer interface with Tyr (orange) and Ala (violet) residues shown as sticks on the Jα-helices.

Figure 9

SAXS of RsLOV in dark and light states. (A) Fits of scaled theoretical monomer (orange) and dimer (blue) scattering curves generated with CRYSOL for the dark (black) and (B) light (gray) state RsLOV (5 mg/mL data). EOM fits generated with RANCH13/GAJOE13 of the monomer were generated from the truncated pdb file of the dimer with only the A subunit. Light state fit composed of 70% EOM monomer model and 30% dimer (lime). (C) Kratky plot of dark and light state RsLOV data of 10 and 5 mg/mL samples. (D) Scattering envelope models based on the 5 mg/mL SAXS data of dark state RsLOV (top, cyan) and light state RsLOV (bottom: green), superimposed with the crystal structure surface of RsLOV, as a complete dimer or truncated to a monomer.

Figure 10

Analogy between RsLOV and sensory Rhodopsin. Left: Structure of RsLOV dimer (violet) overlaid on the dimer of sensory rhodopsin II in complex with helical transducer (pale green, PDB code 1H2S). Right: Overlay of the monomers of RsLOV and sensory rhodopsin II depicted in ribbon with the C-terminal helices aligned and colored darker than the PAS core and transducer helix bundle.