Research Interests: The Lagarias Laboratory
Phytochromes and Their Distribution
Phytochromes were first discovered in flowering plants, but are also widely distributed in other photosynthetic eukaryotes such as mosses, ferns and gymnosperms. Consisting of a large protein, ~1100 amino acids in length, to which a blue-colored bilin pigment is covalently attached, the overall structure of phytochromes has been strongly preserved throughout plant evolution (for overview see Rockwell et al, 2006). Eukaryotic phytochromes are homodimeric chromoproteins. The bilin pigment is associated with a highly conserved 60 kDa N-terminal ‘photosensory input’ domain, whereas the less compact 55 kDa ‘regulatory output’ domain specifies the high-affinity subunit-subunit interaction (Figure 1). The regulatory domain also possesses two subdomains that are crucial for transducing red (R) and far-red (FR) light signals. These include a PAS-related domain, a domain containing two PAS repeats (PASA and PASB), which are found on numerous eukaryotic and prokaryotic regulatory proteins, and a histidine kinase-related domain (HKRD) similar to the ATP-binding and histidine phosphotransferase subdomains found on bacterial two-component sensor proteins.
Figure 1. The structure of eukaryotic phytochromes. Phytochromes from photosynthetic eukaryotes are large proteins consisting of a highly conserved photosensory domain with a covalently attached phytobilin (shown here attached to cysteine residue 322 for oat phytochrome A). Biochemical studies have shown that significant conformational changes occur in the phytochrome protein upon light excitation - changes that have been detected by biochemical probes of protein conformation (i.e. proteases, protein kinases and monoclonal antibodies).
The first complete cyanobacterial genome, that of Synechocystis sp. PCC6803 obtained by the Kazusa DNA Research Institute (http://www.kazusa.or.jp/cyano/cyano.html), revealed the presence of a bonafide phytochrome that was named cyanobacterial phytochrome 1 or Cph1 (see Yeh et al, 1997). Cph1-related proteins are ubiquitously distributed amongst oxygenic photosynthetic bacteria. Like eukaryotic phytochromes, Cph1s possess highly conserved photosensory domains to which the linear tetrapyrrole pigment phycocyanobilin (PCB) is bound. By contrast with plant phytochromes, the two PAS repeats are missing in Cph1s. Figure 3 compares the overall protein architectures of eukaryotic phytochromes and Cph1. Based on biochemical and protein sequence comparisons, the 'photosensory domain' of eukaryotic phytochromes is comprised of four subdomains, named P1 through P4. This figure shows that Cph1 is missing the P1 domain found on eukaryotic phytochromes. Recent investigations have established that the P3 GAF subdomain is sufficient for bilin binding and thus comprises the bilin lyase domain (BLD).
Figure 2. The structure of the extended phytochrome family. The distinguishing structural feature of the extended phytochrome family is the tetrapyrrole-binding GAF domain, which is often associated with transmitter-like histidine kinase domains (HKD) found on bacterial two-component sensor molecules. Proteins with BLD-related GAF domains can be categorized into four families – eukaryotic phytochromes (Phy), cyanobacterial phytochrome 1 (Cph1), cyanobacterial phytochrome 2 (Cph2) and phytochrome-related (Phr) families. GAF domains, which are present in vertebrate cGMP-specific phosphodiesterases, in cyanobacterial adenylate cyclases and in the formate hydrogen lyase transcription activator FhlA, and the structurally related PAS domains found in a large family of transcriptional regulatory proteins including the period clock (PER) protein, the aromatic hydrocarbon receptor nuclear translocator (ARNT) and single minded (SIM) of Drosophila, typically occur in multiple copies in members of the extended phytochrome family.
Since 1996, phytochrome-related protein genes have been identified in many nonphotosynthetic eubacterial and fungal species. Those with protein architectures very similar to Cph1 include bacteriophytochrome (BphP) and fungal phytochrome (Fph) families (Figure 2). While BphP and Fph proteins all lack the invariant cysteine residue to which the phytobilin pigment of phytochromes is covalently attached, many have been shown to form photoactive complexes with biliverdin and are thus thought to function as photoreceptors. Cph1, BphPs and Fphs all lack the PAS domains found on plant phytochromes that are inserted region between the photosensory P1-P4 input domains and the regulatory HKRD at phytochrome's C-terminus.
In addition to Phy and Cph1/BphP/Fph subfamilies, two other phytochrome-related protein families have proliferated in cyanobacteria - Cph2 and Phytochrome-related (Phr) families. Cph2s possess bilin-binding GAF domains quite similar to those of plant and Cph1 phytochromes and function as covalently bound R/FR switchable biliprotein photosensors. As shown in Figure 3, Cph2s typically possess a tandem repeat of up to five GAF domains and a C-terminal HKD. The most N-terminal GAF domain of all Cph2s has been established to be the site where the photochromic bilin prosthetic group is associated. Cph2s invariably possess additional more diverged GAF domains that may be site for additional tetrapyrrole ligands to bind. Members of the Phr family possess more diverged GAF domains that are associated a wide variety of protein modules found in bacterial signaling proteins. These include the cyanobacteriochrome family, which include red/green, green/red and blue/green switchable biliproteins with diverse regulatory activities (see Ikeuchi & Ishizuka, 2008).