Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

Singh, Rajkumar; Kraft, Christian; Jaiswal, Rahul; Sejwal, Kushal; Kasaragod, Vikram Babu; Kuper, Jochen; Bürger, Jörg; Mielke, Thorsten; Luirink, Joen; Bhushan, Shashi

doi:10.1074/jbc.m113.506634

Cited by 40 publications

(61 citation statements)

References 54 publications

(35 reference statements)

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“…This occurs perhaps post-translationally, but we have no evidence that bears on the question. Based upon recent structural studies showing that SecA binds to the exit tunnel of the ribosome [47; 48], it is possible that SecA interacts with the nascent chain in a co-translational manner. If insertion is post-translational, one possibility is that the TM segment first partitions into the membrane interface followed by SecA-guided TM insertion and C-terminal transport through the SecYEG translocation channel.…”

Section: Discussionmentioning

confidence: 99%

SecA Drives Transmembrane Insertion of RodZ, an Unusual Single-Span Membrane Protein

Rawat

Zhu

Lindner

et al. 2015

Journal of Molecular Biology

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The transmembrane (TM) helices of most type II single-span membrane proteins (S-SMPs) of Escherichia coli occur near the N-terminus, where the cell’s targeting mechanisms can readily identify it as it emerges from the ribosome. But the TM helices of a few S-SMPs, such as RodZ, occur a hundred or more residues downstream from the N-terminus, which raises fundamental questions about targeting and assembly. Because of RodZ’s novelty and potential usefulness for understanding TM helix insertion in vivo, we examined its membrane targeting and assembly. We used RodZ constructs containing immuno tags before the TM domain to assess membrane insertion using Proteinase K digestion. We confirmed the Nin-Cout (Type II) topology of RodZ and established the absence of a targeting signal other than the TM domain. RodZ was not inserted into the membrane under SecA-depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. Insertion failed when the transmembrane proton gradient was abolished with CCCP. Insertion also failed when RodZ was expressed in SecE-depleted E. coli, indicating that the SecYEG translocon is required for RodZ assembly. Protease accessibility assays of RodZ in other E. coli depletion strains revealed that insertion is independent of SecB, YidC, and SecD/F. Insertion was found to be only weakly dependent on the signal recognition particle (SRP) pathway: insertion was weakly dependent on the Ffh but independent of FtsY. We conclude that membrane insertion of RodZ requires only the SecYEG translocon, the SecA ATPase motor, and the transmembrane proton motive force (PMF).

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Section: Discussionmentioning

confidence: 99%

SecA Drives Transmembrane Insertion of RodZ, an Unusual Single-Span Membrane Protein

Rawat

Zhu

Lindner

et al. 2015

Journal of Molecular Biology

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“…The binding site for SecA on the ribosome includes ribosomal protein uL23, which is located adjacent to the opening of the polypeptide exit channel (Huber et al, 2011). Binding is mediated by the N-terminal α-helix of SecA and the N-terminal portion of the HSD (Huber et al, 2011; Singh et al, 2014). The structure of the SecA-ribosome complex was recently determined at medium resolution (∼11 Å) by cryo-electron microscopy (Singh et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…Binding is mediated by the N-terminal α-helix of SecA and the N-terminal portion of the HSD (Huber et al, 2011; Singh et al, 2014). The structure of the SecA-ribosome complex was recently determined at medium resolution (∼11 Å) by cryo-electron microscopy (Singh et al, 2014). However, the molecular mechanism governing the recognition of nascent substrate proteins is not known.…”

Section: Introductionmentioning

confidence: 99%

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

Jamshad

Knowles

White

et al. 2018

Preprint

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37SecA is an evolutionarily conserved and essential ATPase that is required for the 38 translocation of a subset of proteins across the cytoplasmic membrane in bacteria. SecA can 39 recognise proteins that are destined for translocation as they are still being synthesised in 40 order to deliver them to the membrane-bound Sec machinery. However, the mechanism of 41 cotranslational substrate recognition is not well defined. In E. coli, SecA contains a relatively 42 long C-terminal tail (CTT), which consists of a small metal-binding domain (MBD) that is 43 attached to the C-terminus of the catalytic core by a flexible linker (FLD). In this study, we 44 investigated the role of the CTT in the interaction of SecA with the ribosome and nascent 45 polypeptides. Previous work indicates that the CTT is required for interaction with the 46 molecular chaperone SecB. However, phylogenetic analysis suggested that the CTT (and the 47 MBD in particular) has an additional function. Binding experiments indicated that the CTT 48 interacts with 70S ribosomes, and disruption of the entire CTT moderately reduced the 49 affinity of SecA for ribosomes. However, disruption of the MBD alone significantly 50 increased the affinity of SecA for ribosomes and inhibited the interaction of SecA with 51 substrate protein, suggesting that the FLD affects the conformation of SecA. 52 Photocrosslinking and mass spectrometry indicated that the FLD is bound at the site where 53 SecA binds to substrate proteins. Structural analysis by x-ray crystallography and small-angle 54 x-ray scattering (SAXS) provided insight into how the CTT influences the structure of SecA 55 in solution. Finally, site-specific crosslinking experiments indicated that binding to nascent 56 substrate protein affects the conformation of SecA. Taken together, our results suggest that 57 the CTT regulates the ability of SecA to interact with substrate proteins.58 105 involved in binding of SecA to ribosomes, which we confirmed using ribosome 106 cosedimentation and chemical crosslinking approaches. Strikingly, disruption of the MBD 107 alone or the entire CTT had opposing effects on multiple different activities of SecA, 108 6 suggesting that the CTT affects conformation of the catalytic core. Mass spectrometry, x-ray 109 crystallography, and small-angle x-ray scattering experiments indicated that the FLD is 110 bound in the substrate binding groove and affects the conformation of the PPXD. Finally, site 111 specific chemical crosslinking suggested that binding of the MBD to the ribosome allows 112 full-length SecA to interact with nascent substrate proteins. Taken together, our results 113 provide insight into the molecular mechanism underlying nascent substrate recognition by 114 SecA. 115 116 Results 117 Evolutionary distribution of the MBD of SecA. To investigate the evolutionary 118 distribution of the CTT, we analysed the sequences of 156 SecA proteins from bacterial 119 species in 155 phylogenetic families using ClustalOmega (McWilliam et al., 2013). The 120 phylogenetic tr...

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“…The interaction is at the busy exit site, where it must compete for access to the nascent chain with SRP (36, 42). This raised interesting questions about the role of SecA in cotranslational translocation, which the current work explores.…”

Section: Introductionmentioning

confidence: 99%

“…For instance, SecA dimer dissociation (47, 48) could promote ATP activation and intercalation of the preprotein (49); interestingly, the structure of the ribosome bound to SecA reveals that both one and two copies can associate (42). Additionally, the formation of a strong interaction with SecB about the unfolded mature regions night help to ensure its efficient transport (Fig.…”

Section: Introductionmentioning

confidence: 99%

SecA—a New Twist in the Tale

Collinson

2017

J Bacteriol

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A paper published in this issue of the Journal of Bacteriology (D. Huber, M. Jamshad, R. Hanmer, D. Schibich, K. Döring, I. Marcomini, G. Kramer, and B. Bukau, J Bacteriol 199:e0622-16, 2017, https://doi.org/10.1128/JB.00622-16) provides us with a timely reminder that all is not as clear as we had previously thought in the general bacterial secretion system. The paper describes a new mode of secretion through the Sec system—“uncoupled cotranslocation”—for the passage of proteins across the bacterial inner membrane and suggests that we might rethink the nature and mechanism of the targeting and transport steps toward protein export.

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Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

Cited by 40 publications

References 54 publications

SecA Drives Transmembrane Insertion of RodZ, an Unusual Single-Span Membrane Protein

SecA Drives Transmembrane Insertion of RodZ, an Unusual Single-Span Membrane Protein

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

SecA—a New Twist in the Tale

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