Structures, Mechanisms and Biogenesis of Biliproteins

Bilins are biological pigments that play important roles in many light-dependent processes in both photosynthetic and non-photosynthetic organisms. Despite the extensive studies in recent decades, many important questions regarding the light signaling mechanisms of bilin-based photoreceptors and biogenesis of light harvesting phycobiliproteins remain unanswered. Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. In response to light, bacteriophytochromes undergo autophosphorylation at the histidine kinase (HK) domain, as the first step of the predominant two-component signal-transduction mechanism in bacteria. However, the molecular basis and directionality of the autophosphorylation process (cis; intra-subunit or trans; inter-subunit) within the dimeric HK remain unknown. In this dissertation we present a complementary rescue strategy based on loss-of-function mutants of conserved His and catalytic Asn residues in the HK domain of two tandem bacteriophytochromes from the photosynthetic bacterium Rhodopseudomonas palustris RpBphP2 and RpBphP3 to distinguish the mode of phosphorylation between cis and trans by HK assay using phos-tag gels. Our results unambiguously show that these bacteriophytochromes undergo trans phosphorylation individually and as two tandem heterodimers. The crystal structure of the isolated HK domain of the full-length RpBphP2 and the modelled full-length structure allowed to understand the catalytic mechanism of the HK trans-autophosphorylation in bacteriophytochromes. Extensive bilin pigment diversity of the light harvesting complexes in marine Synechococcus significantly contributes to their widespread abundance in the ocean. Such diversity depends on bilin lyases that attach chemically distinct chromophores to the phycobiliproteins. A distinct group of the E/F type bilin lyases acquired additional abilities to isomerize bilins, which further expanded the pigment repertoire for light absorption. Despite recent structural studies on a representative CpcE/F, the overall architecture, active site geometry and reaction mechanism remain unresolved for the E/F family of bilin lyases. In this dissertation we report the crystal structure at 2.5 Å resolution of MpeQ, a newly identified bilin lyase-isomerase that plays important roles in type IV chromatic acclimation. The single-chain -solenoid structure of MpeQ reveals a large active-site chamber in a “question-mark”-shaped protein architecture. Using site-directed mutagenesis, we identified key residues responsible for the lyase and isomerase activities of MpeQ. Our structural analyses have revealed a nucleophilic tyrosine within a catalytic triad in several bilin lyases, suggesting a shared reaction mechanism among bilin lyases of distinct protein scaffolds. We also propose that the isomerase activity of MpeQ is resulted from steric incompatibility between the active site geometry and the A-ring conformation of the bilin substrate.