Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Gao, Feng; Kight, Alicia; Henderson, Rory; Jayanthi, Srinivas; Patel, Parth; Murchison, Marissa; Sharma, Priyanka; Goforth, Robyn L.; Kumar, Thallapuranam Krishnaswamy Suresh; Henry, Ralph; Heyes, Colin D.

doi:10.1074/jbc.m114.624346

Cited by 22 publications

(50 citation statements)

References 49 publications

(68 reference statements)

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“…The mutants produced were S54C in the N-domain and K407C in the M-domain of cpSRP54 (cpSRP54 S54C/K407C ). The peak in the smFRET histogram is wider than expected from a single state, similar to our previous report for cpSRP43 (27), indicating that at least two conformational states are present (Fig. 2).…”

Section: The Orientation Of the M-domain Varies In Different Srp54s/ffhssupporting

confidence: 90%

“…This study, along with recent findings for cpSRP43 (27), reveals the cpSRP system is characterized by a large amount of structural heterogeneity. While cpSRP43 prepares an energetic funnel favoring LHCP interactions downstream from cpSRP formation, cpSRP54 appears to use various domain orientations to allow proper positioning of its domains for LHCP loading.…”

Section: Discussionsupporting

confidence: 78%

“…Minimization and equilibration of the system was carried out in the NPT ensemble using NAMD 2.9 and the CHARMM27 or CHARMM36 force fields (50)(51)(52). The protein-solvent system was equilibrated as described previously in Gao et al (27). To monitor the stability of the proteins and to evaluate structurally variable regions, simulations of 100 ns using a 2-fs time-step were performed.…”

Section: Simulationmentioning

confidence: 99%

“…A double-cysteine mutant of cpSRP54 (cpSRP54 S54C/K407C ) was sitespecifically labeled using Alexa488-C5-maleimide and Alexa594-C5-maleimide (Life Technologies), and the single-molecule (smFRET) experiments were performed in HKMK buffer (10 mM HEPES, 10 mM MgCl 2 , and 100 mM KCl, pH 8.0) and carried out using a MicroTime 200 microscope controlled by SymPhoTime software (PicoQuant, Berlin, Germany), collecting fluorescence photons on a photon-by-photon basis, as previously described in Gao et al (27). Briefly, a water immersion objective with an N.A.…”

Section: Single-molecule Fretmentioning

confidence: 99%

“…Atomic level details of LHCP targeting by cpSRP, however, remain elusive. Recent work has demonstrated significant conformational dynamics in cpSRP43 that support modulation of downstream interactions (27). However, little structural information exists to explain how cpSRP54 is able to function in the classical, cotranslational signaling process as well as in the nonclassical, posttranslational targeting of LHCP.…”

Section: Introductionmentioning

confidence: 99%

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Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle

Henderson

Gao

Jayanthi

et al. 2016

Biophysical Journal

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Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico threedimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Fö rster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post-and cotranslational signaling processes.

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Section: The Orientation Of the M-domain Varies In Different Srp54s/ffhssupporting

confidence: 90%

Section: Discussionsupporting

confidence: 78%

Section: Simulationmentioning

confidence: 99%

Section: Single-molecule Fretmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle

Henderson

Gao

Jayanthi

et al. 2016

Biophysical Journal

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Distinct encounter complexes of PAI-1 with plasminogen activators and vitronectin revealed by changes in the conformation and dynamics of the reactive center loop

et al. 2015

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Plasminogen activator inhibitor-1 (PAI-1) is a biologically important serine protease inhibitor (serpin) that, when overexpressed, is associated with a high risk for cardiovascular disease and cancer metastasis. Several of its ligands, including vitronectin, tissue-type and urokinase-type plasminogen activator (tPA, uPA), affect the fate of PAI-1. Here, we measured changes in the solvent accessibility and dynamics of an important unresolved functional region, the reactive center loop (RCL), upon binding of these ligands. Binding of the catalytically inactive S195A variant of tPA to the RCL causes an increase in fluorescence, indicating greater solvent protection, at its C-terminus, while mobility along the loop remains relatively unchanged. In contrast, a fluorescence increase and large decrease in mobility at the N-terminal RCL is observed upon binding of S195A-uPA to PAI-1. At a site distant from the RCL, binding of vitronectin results in a modest decrease in fluorescence at its proximal end without restricting overall loop dynamics. These results provide the new evidence for ligand effects on RCL conformation and dynamics and differences in the Michaelis complex with plasminogen activators that can be used for the development of more specific inhibitors to PAI-1. This study is also the first to use electron paramagnetic resonance (EPR) spectroscopy to investigate PAI-1 dynamics.Significance: Balanced blood homeostasis and controlled cell migration requires coordination between serine proteases, serpins, and cofactors. These ligands form noncovalent complexes, which influence the outcome of protease inhibition and associated physiological processes. This study reveals differences in binding via changes in solvent accessibility and dynamics within these Abbreviations: DTT, dithiothreitol; EPR, electron paramagnetic resonance; ki, second-order rate of inhibition; k lim , limiting rate of insertion; MTSL, 2,5-dihydro-2,

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Absence of photosynthetic state transitions in alien chloroplasts

Yeates

Zubko

Ruban

2019

Planta

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Main conclusion The absence of state transitions in a Nt ( Hn ) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. Abstract The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane ( Hyoscyamus niger ) chloroplasts function on the nuclear background of a tobacco ( Nicotiana tabacum ). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions—a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional ‘LHCII’ band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions—the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants. Electronic supplementary material The online version of this article (10.1007/s00425-019-03187-2) contains supplementary material, which is available to authorized users.

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Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Cited by 22 publications

References 49 publications

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle

Distinct encounter complexes of PAI-1 with plasminogen activators and vitronectin revealed by changes in the conformation and dynamics of the reactive center loop

Absence of photosynthetic state transitions in alien chloroplasts

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