5 research outputs found
Structures, Mechanisms and Biogenesis of Biliproteins
No full textBilins 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
Dimer asymmetry and light activation mechanism in brucella blue-light sensor histidine kinase
Get PDFThe ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus. Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain. IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Fernandez, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Shin, Heewhan. University of Illinois; Estados UnidosFil: Sycz, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Gunawardana, Semini. University of Illinois; Estados UnidosFil: Kumarapperuma, Indika. University of Illinois; Estados UnidosFil: Aragón Paz, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Otero, Lisandro Horacio. Plataforma Argentina de Biología Estructural y Metabolómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cerutti, Maria Laura. Plataforma Argentina de Biología Estructural y Metabolómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ren, Zhong. University of Illinois; Estados UnidosFil: Klinke, Sebastian. Plataforma Argentina de Biología Estructural y Metabolómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Yang, Xiaojing. University of Illinois; Estados UnidosFil: Goldbaum, Fernando Alberto. Plataforma Argentina de Biología Estructural y Metabolómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin
Crystal-on-crystal chips for in situ serial diffraction at room temperature
No full textRecent developments in serial crystallography at X-ray free electron lasers (XFELs) and synchrotrons have been driven by two scientific goals in structural biology - first, static structure determination from nano or microcrystals of membrane proteins and large complexes that are difficult for conventional cryocrystallography, and second, direct observations of transient structural species in biochemical reactions at near atomic resolution. Since room-temperature diffraction experiments naturally demand a large quantity of purified protein, sample economy is critically important for all steps of serial crystallography from crystallization, crystal delivery to data collection. Here we report the development and applications of "crystal-on-crystal" devices to facilitate large-scale in situ serial diffraction experiments on protein crystals of all sizes - large, small or microscopic. We show that the monocrystalline quartz as a substrate material prevents vapor loss during crystallization and significantly reduces background X-ray scattering. These devices can be readily adopted at XFEL and synchrotron beamlines, which enable efficient delivery of hundreds to millions of crystals to the X-ray beam, with an overall protein consumption per dataset comparable to that of cryocrystallography
Crystal structure and molecular mechanism of an E/F type bilin lyase-isomerase
No full textInternational audienceChromophore attachment of the light-harvesting apparatus represents one of the most important post-translational modifications in photosynthetic cyanobacteria. Extensive pigment diversity of cyanobacteria critically depends on bilin lyases that covalently attach chemically distinct chromophores to phycobiliproteins. However, how bilin lyases catalyze bilin ligation reactions and how some lyases acquire additional isomerase abilities remain elusive at the molecular level. Here, we report the crystal structure of a representative bilin lyase-isomerase MpeQ. This structure has revealed a “question-mark” protein architecture that unambiguously establishes the active site conserved among the E/F-type bilin lyases. Based on structural, mutational, and modeling data, we demonstrate that stereoselectivity of the active site plays a critical role in conferring the isomerase activity of MpeQ. We further advance a tyrosine-mediated reaction scheme unifying different types of bilin lyases. These results suggest that lyases and isomerase actions of bilin lyases arise from two coupled molecular events of distinct origi
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Light-induced protein structural dynamics in bacteriophytochrome revealed by time-resolved x-ray solution scattering
Get PDFBacteriophytochromes (BphPs) are photoreceptors that regulate a wide range of biological mechanisms via red light–absorbing (Pr)–to–far-red light–absorbing (Pfr) reversible photoconversion. The structural dynamics underlying Pfr-to-Pr photoconversion in a liquid solution phase are not well understood. We used time-resolved x-ray solution scattering (TRXSS) to capture light-induced structural transitions in the bathy BphP photosensory module of Pseudomonas aeruginosa. Kinetic analysis of the TRXSS data identifies three distinct structural species, which are attributed to lumi-F, meta-F, and Pr, connected by time constants of 95 μs and 21 ms. Structural analysis based on molecular dynamics simulations shows that the light activation of PaBphP accompanies quaternary structural rearrangements from an “II”-framed close form of the Pfr state to an “O”-framed open form of the Pr state in terms of the helical backbones. This study provides mechanistic insights into how modular signaling proteins such as BphPs transmit structural signals over long distances and regulate their downstream biological responses
