Large conformational changes of a highly dynamic pre-protein binding domain in SecA (original) (raw)
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Using a Low Denaturant Model To Explore the Conformational Features of Translocation-Active SecA
Biochemistry, 2012
The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive posttranslational secretion of pre-proteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, 'closed' state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more 'open', has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1), and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon. Protein secretion is an essential process in all forms of life. In gram-negative bacteria, newly synthesized proteins destined for integration into membranes or secretion into the extracellular milieu predominantly traverse the secretory (Sec) pathway (1, 2). Most preproteins are targeted to the Sec pathway by a cleavable N-terminal signal sequence and by the peripheral membrane motor protein, SecA. E. coli SecA is a large, dynamic 102 kDa protein that forms homodimers and interacts with many different players during the translocation cycle, including pre-proteins, SecB, membrane, and the SecYEG translocon (3). The cytosolic chaperone SecB binds to a subset of pre-proteins, keeping them in a
2019
The Sec translocon is a highly conserved membrane complex for transport of polypeptides across, or into, lipid bilayers. In bacteria, the core protein-channel SecYEG resides in the inner-membrane, through which secretion is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the dynamic state of SecYEG throughout the hydrolytic cycle of SecA. We show that the SecYEG channel fluctuates between open and closed states faster (∼20-fold during transport) than ATP turnover; while the nucleotide status of SecA modulates the rates of opening and closure. Interestingly, a SecY variant (PrlA4), exhibiting faster protein transport, but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore; thereby improving the efficiency of translocation. Thus, contrary to prevailing structure-based models, SecYEG plays an integral part in the translocation mechanism through dynamic allosteric couplin...
A molecular switch in SecA protein couples ATP hydrolysis to protein translocation
Molecular …, 1999
SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the Ndomain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the Ndomain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.
Journal of Peptide Science, 2011
Bacteria employ the SecA motor protein to push unfolded proteins across the cytoplasmic membrane through the SecY protein-conducting channel complex. The crystal structure of the SecA-SecY complex shows that the intramolecular regulator of ATPase1 (IRA1) SecA domain, made up of two helices and the loop between them, is partly inserted into the SecY conducting channel, with the loop between the helices as the main functional region. A computational analysis suggested that the entire IRA1 domain is structurally autonomous, and was the basis to synthesize peptide analogues of the SecA IRA1 loop region, to the aim of investigating its conformational preferences. Our study indicates that the loop region populates a predominantly flexible state, even in presence of structuring agent. This provides indirect evidence that the SecA loop-SecY receptor docking involves loop-mediated opening of the SecY channel.
Mapping polypeptide interactions of the SecA ATPase during translocation
Proceedings of the National Academy of Sciences, 2009
Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a “clamp” of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA's clamp and moves in an extended conformation through the clamp into the SecY channel. These results ...
Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation
The Sec translocon is a transmembrane assembly highly conserved among all forms of life as the principal route for transport of polypeptides across or into lipid bilayers. In bacteria translocation is driven by allosteric communication between the membrane pore SecYEG and the associated SecA ATPase. Using time-resolved single molecule fluorescence we reveal that slow conformational changes associated with SecA ATPase (~ 6 s-1) modulate fast opening and closure of the SecY pore (~ 175 s-1). Such mismatch of timescales is not compatible with direct coupling between SecA and SecYEG and the power stroke mechanism. A dynamic allosteric model in which SecA ATPase cycle controls energy landscape for SecY pore opening is proposed and consistent with the Brownian-ratchet mechanism. Analysis of structures and molecular dynamics trajectories identified key molecular interactions involved in the mechanism. This dynamic allostery may be common among motor ATPases that drive conformational changes in molecular machines.
Dynamic action of the Sec machinery during initiation, protein translocation and termination
eLife, 2018
Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.