Light Signaling Mechanism of Two Tandem Bacteriophytochromes

Abstract

RpBphP2 and RpBphP3, two tandem bacteriophytochromes from the photosynthetic bacterium R. palustris, share high sequence identity but exhibit distinct photoconversion behavior. Unlike the canonical RpBphP2, RpBphP3 photoconverts to an unusual nearred-absorbing (Pnr) state; both are required for synthesis of light harvesting complexes under low-light conditions. Here we report the crystal structures of the photosensory core modules of RpBphP2 and RpBphP3. Despite different quaternary structures, RpBphP2 and RpBphP3 adopt nearly identical tertiary structures. The RpBphP3 structure reveals “tongue-and-groove” interactions at the interface between the GAF and PHY domains. A single mutation in the PRxSF motif at the GAF-PHY interface abolishes light-induced formation of the Pnr state in RpBphP3, possibly due to altered structural rigidity of the chromophore-binding pocket. Structural comparisons suggest that long-range signaling involves structural rearrangement of the helical spine at the dimer interface. These structures together with mutational studies provide insights into photoconversion and long-range signaling mechanism in phytochromes.

The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers

“…The arrangement of two protein subunits in the crystals is parallel in the case of PaBphP and anti-parallel in the case of Cph1. A parallel arrangement was also found for the CBD (24) and the PCM (25) of DrBphP, the PCMs of plant phytochrome (26) and of RpBphP2 from Rhodopseudomonas palustris (27), and other CBD or PCM structures (28). Anti-parallel orientations were observed in crystals of Cph2 (14) and of RpBphP3 from R. palustris (27).…”

“…Buffer conditions and pH during crystal growth were also similar. Another anti-parallel arrangement, but within a different crystal packing, has recently been described for the PCM of RpBphP3 from R. palustris in the Pr form (27). Of the 13 available PCM crystal structures of different phytochromes in different photochromic states, 10 are in the Pr state, and three of these assume an anti-parallel arrangement, whereas of three Pfr state or Pfr-like structures, two exhibit a parallel subunit arrangement, the only exception being R. palustris phytochrome RpBphP1 (28), which is not an HK.…”

“…2, A, C and D, and 4) resemble the known PCM structures of canonical phytochromes, i.e. the prototypical phytochromes Cph1 (23); PhyB from Arabidopsis (25); RpBphP2 and RpBphP3 (27); DrBphP (25,29); and the bathy phytochromes PaBphP (21) and R. palustris RpBphP1 (28). This includes the figure of eight knot formed by PAS and GAF domains (22), a long helix connecting the GAF and PHY domains, a long helix at the C terminus that connects with the HK, and the "tongue of the PHY domain," a structure that folds back on and closes the chromophore pocket that is largely formed by the GAF domain ( Fig.…”

“…6, C and E; Table 3), consistent with the idea that this Pro is required for Pr-to-Pfr conversion in these phytochromes by a mechanism that is similar but not necessarily identical to the one suggested here for Agp1. In R. palustris RpBphP3, the homologous residue is a Thr that is 4.5 Å away from ring A of the BV chromophore (Table 3) (27). This phytochrome, however, converts into an atypical near-red light-absorbing state photoproduct with a shorter wavelength absorbance maximum, which according to our hypothesis is due to the lack of steric strain in the ring A region in the Pr state that would be required for photoconversion into a Pfr state, and therefore it results in the photo-activated state of RpBphP3 thermally relaxing via a different pathway into the near-red light-absorbing state.…”

“…This is due to a kink around position 308, the transition point between both domains. A different bending has also been described for long GAF-PHY connecting helices in other phytochrome structures (27,30). Because structural changes in the long helices connecting the GAF and the PHY domain as well as the PHY and the HK domain are thought to be crucial for the propagation of a signal from the sensory domain to the signal output domain (54), we compared the relative orientations of (24) and is different from the BV-like structure also shown in A.…”