scholarly journals Allosteric Response and Substrate Sensitivity in Peptide Binding of the Signal Recognition Particle

2014 ◽  
Vol 289 (44) ◽  
pp. 30868-30879 ◽  
Author(s):  
Connie Y. Wang ◽  
Thomas F. Miller
Keyword(s):  
Single Molecule ◽  
Protein Targeting ◽  
Substrate Binding ◽  
Body Movement ◽  
Conformational Stability ◽  
Conformational Dynamics ◽  
Signal Recognition Particle ◽  
Central Component ◽  
Signal Recognition ◽  
Dynamics Simulations

We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway.

scholarly journals Receptor compaction and GTPase movements drive cotranslational protein translocation

2020 ◽  
Author(s):  
Jae Ho Lee ◽  
SangYoon Chung ◽  
Yu-Hsien Hwang Fu ◽  
Ruilin Qian ◽  
Xuemeng Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Biological Regulation ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Cause Of Failure ◽  
Distal Site

AbstractSignal recognition particle (SRP) is a universally conserved targeting machine that couples the synthesis of ~30% of the proteome to their proper membrane localization1,2. In eukaryotic cells, SRP recognizes translating ribosomes bearing hydrophobic signal sequences and, through interaction with SRP receptor (SR), delivers them to the Sec61p translocase on the endoplasmic reticulum (ER) membrane1,2. How SRP ensures efficient and productive initiation of protein translocation at the ER is not well understood. Here, single molecule fluorescence spectroscopy demonstrates that cargo-loaded SRP induces a global compaction of SR, driving a >90 Å movement of the SRP•SR GTPase complex from the vicinity of the ribosome exit, where it initially assembles, to the distal site of SRP. These rearrangements bring translating ribosomes near the membrane, expose conserved Sec61p docking sites on the ribosome and weaken SRP’s interaction with the signal sequence on the nascent polypeptide, thus priming the translating ribosome for engaging the translocation machinery. Disruption of these rearrangements severely impairs cotranslational protein translocation and is the cause of failure in an SRP54 mutant linked to severe congenital neutropenia. Our results demonstrate that multiple largescale molecular motions in the SRP•SR complex are required to drive the transition from protein targeting to translocation; these post-targeting rearrangements provide potential new points for biological regulation as well as disease intervention.

scholarly journals Sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient protein targeting

2018 ◽  
Vol 115 (24) ◽  
pp. E5487-E5496 ◽  
Author(s):  
Jae Ho Lee ◽  
Sowmya Chandrasekar ◽  
SangYoon Chung ◽  
Yu-Hsien Hwang Fu ◽  
Demi Liu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Targeted Delivery ◽  
Protein Targeting ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Optimal Conformation ◽  
Sequential Mechanism ◽  
Sequential Activation ◽  
Sense And Respond

Signal recognition particle (SRP) is a universally conserved targeting machine that mediates the targeted delivery of ∼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective protein targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective protein targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.

SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 101-104 ◽  
Author(s):  
Jan Timo Grotwinkel ◽  
Klemens Wild ◽  
Bernd Segnitz ◽  
Irmgard Sinning
Keyword(s):  
Ribosomal Rna ◽  
Protein Targeting ◽  
Rna Binding ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Structural Basis ◽  
Signal Recognition ◽  
Rna Remodeling ◽  
Srp Rna ◽  
Arginine Rich Motif

The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

scholarly journals Real-time observation of signal recognition particle binding to actively translating ribosomes

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Thomas R Noriega ◽  
Jin Chen ◽  
Peter Walter ◽  
Joseph D Puglisi
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Initial Step ◽  
Signal Recognition ◽  
Fluorescence Measurements ◽  
Nascent Chain ◽  
Time Observation

The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

scholarly journals Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking

2019 ◽  
Vol 5 (12) ◽  
pp. eaay5912 ◽  
Author(s):  
Devynn M. Wulstein ◽  
Kathryn E. Regan ◽  
Jonathan Garamella ◽  
Ryan J. McGorty ◽  
Rae M. Robertson-Anderson
Keyword(s):  
Transport Properties ◽  
Single Molecule ◽  
Intracellular Transport ◽  
Conformational Stability ◽  
Conformational Dynamics ◽  
Single Mode ◽  
Dna Molecules ◽  
Linear Dna ◽  
Linear Chains ◽  
Anomalous Dynamics

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

Regulation by the ribosome of the GTPase of the signal-recognition particle during protein targeting

Nature ◽  
1996 ◽  
Vol 381 (6579) ◽  
pp. 248-251 ◽  
Author(s):  
Gerald Bacher ◽  
Henrich Lütcke ◽  
Berit Jungnickel ◽  
Tom A. Rapoport ◽  
Bernhard Dobberstein
Keyword(s):  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Signal Recognition

scholarly journals In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host

2002 ◽  
Vol 184 (12) ◽  
pp. 3260-3267 ◽  
Author(s):  
R. Wesley Rose ◽  
Mechthild Pohlschröder
Keyword(s):  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sequence Comparisons ◽  
Protein Insertion ◽  
Native Host

ABSTRACT The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.

scholarly journals Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

2015 ◽  
Vol 211 (1) ◽  
pp. 91-104 ◽  
Author(s):  
Patrick Kuhn ◽  
Albena Draycheva ◽  
Andreas Vogt ◽  
Narcis-Adrian Petriman ◽  
Lukas Sturm ◽  
...  
Keyword(s):  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Resonance Energy Transfer ◽  
Chain Complex ◽  
Resonance Energy ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Ribosomal Tunnel ◽  
Nascent Chain

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex.

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