Anastassios Economou

Structural Basis for Signal-Sequence Recognition by the Translocase Motor SecA as Determined by NMR

Recognition of signal sequences by cognate receptors controls the entry of virtually all proteins to export pathways. Despite its importance, this process remains poorly understood. Here, we present the solution structure of a signal peptide bound to SecA, the 204 kDa ATPase motor of the Sec translocase. Upon encounter, the signal peptide forms an alpha-helix that inserts into a flexible and elongated groove in SecA. The mode of binding is bimodal, with both hydrophobic and electrostatic interactions mediating recognition. The same groove is used by SecA to recognize a diverse set of signal sequences. Impairment of the signal-peptide binding to SecA results in significant translocation defects. The C-terminal tail of SecA occludes the groove and inhibits signal-peptide binding, but autoinhibition is relieved by the SecB chaperone. Finally, it is shown that SecA interconverts between two conformations in solution, suggesting a simple mechanism for polypeptide translocation.

Bacterial protein secretion through the translocase nanomachine

All cells must traffic proteins across their membranes. This essential process is responsible for the biogenesis of membranes and cell walls, motility and nutrient scavenging and uptake, and is also involved in pathogenesis and symbiosis. The translocase is an impressively dynamic nanomachine that is the central component which catalyses transmembrane crossing. This complex, multi-stage reaction involves a cascade of inter- and intramolecular interactions that select, sort and target polypeptides to the membrane, and use energy to promote the movement of these polypeptides across — or their lateral escape and integration into — the phospholipid bilayer, with high fidelity and efficiency. Here, we review the most recent data on the structure and function of the translocase nanomachine.

The Rhizobium nodulation gene nodO encodes a Ca2(+)-binding protein that is exported without N-terminal cleavage and is homologous to haemolysin and related proteins.

Nodulation and host‐specific recognition of legumes such as peas and Vicia spp. are encoded by the nodulation (nod) genes of Rhizobium leguminosarum biovar viciae. One of these genes, nodO, has been shown to encode an exported protein that contains a multiple tandem repeat of a nine amino acid domain. This domain was found to be homologous to repeated sequences in a group of bacterial exported proteins that includes haemolysin, cyclolysin, leukotoxin and two proteases. These proteins are secreted by a mechanism that does not involve an N‐terminal signal peptide. The NodO protein is present in the growth medium of Rhizobium bacteria induced for nod gene expression, and partial protein sequencing of the purified protein showed that there is no N‐terminal cleavage of the exported protein. It has been suggested that the internally repeated domain of haemolysin may be involved in Ca2(+)‐mediated binding to erythrocytes and we show that the NodO protein can bind 45Ca2+. It is proposed that the NodO protein may interact directly with plant root cells in a Ca2(+)‐dependent way, thereby mediating an early stage in the recognition that occurs between Rhizobium and its host legume.

Following the leader: bacterial protein export through the sec pathway

Significant strides have been made during the past 20 years in our understanding of protein secretion across the bacterial inner membrane. Specialized chaperones select secretory polypeptide chains and usher them to a membrane-embedded preprotein translocase. This unique molecular machine envelops the polymeric substrate and migrates along its length in defined, energy-dependent steps. Consequently, preproteins are gradually pumped into the periplasm where they acquire their native, folded conformation.

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation

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 N‐domain 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 N‐domain and the C‐domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.

Structural Basis for Protein Antiaggregation Activity of the Trigger Factor Chaperone

Introduction Molecular chaperones prevent aggregation and misfolding of proteins in the cellular environment and are thus central to maintaining protein homeostasis. Molecular chaperones are thought to recognize and bind to exposed hydrophobic regions of the unfolded proteins, thereby shielding these regions from the solvent. If unprotected, the proteins would likely aggregate or misfold to bury the hydrophobic residues. Despite the central importance of the binding of chaperones to unfolded proteins, the structural basis of their interaction remains poorly understood. The scarcity of structural data on complexes between chaperones and unfolded proteins is primarily due to technical challenges originating in the size and dynamic nature of these complexes. Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein.Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein. Rationale Recent advances in nuclear magnetic resonance (NMR) and isotope labeling approaches make it possible to study large, dynamic complexes. We used NMR spectroscopy to characterize the binding of the 48-kD unfolded alkaline phosphatase (PhoA) to the 50-kD trigger factor (TF) chaperone. We obtained atomic insight into the dynamic binding and determined the solution structure of PhoA captured in an extended, unfolded state by three TF molecules. Based on our NMR studies, we gained insight into how TF rescues an aggregation-prone protein and how it exerts its unfoldase activity. Results We show that TF uses multiple sites, which are located in two different domains and extend over a distance of ~90 Å, to bind to several regions of the unfolded PhoA that are dispersed throughout its entire length. Three TF molecules are required to interact with the entire length of PhoA, giving rise to a ~200-kD complex in solution. The TF-PhoA interactions are mediated primarily by hydrophobic contacts. TF interacts with PhoA in a highly dynamic fashion, giving rise to a rugged landscape for the free energy of interaction. As the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. The multivalent binding keeps PhoA in an extended, unfolded conformation. Crucially, even the lowest-energy TF-PhoA complex remains rather dynamic with a lifetime of ~20 ms. The structural data of the three TF molecules in complex with different regions of PhoA reveal how the same binding sites within a molecular chaperone can recognize and interact with a large number of substrates with unrelated primary sequences. This promiscuous recognition is further enabled by the notable plasticity of the substrate-binding sites in TF. We finally show that TF in the cytosol prevents aggregation by interacting transiently with the low-populated, aggregation-prone unfolded state of the substrate but acts as a powerful unfoldase when it is bound at the ribosome and thus is colocalized with translating substrate. Conclusion The structural data reveal a multivalent binding mechanism between the chaperone and its protein substrate. This mechanism of binding presents several advantages as it enables chaperones to function as holdases and unfoldases by exerting forces to retain proteins in the unfolded state and at the same time protect them from aggregation by shielding their exposed hydrophobic regions. Given the existence of multiple binding sites in other molecular chaperones, this may present a general mechanism for the action of molecular chaperones. The fast kinetics of substrate binding enables chaperones to interact with transiently exposed, aggregation-prone regions of unstable proteins in the cytosol, thereby preventing their aggregation and increasing their solubility. Nuclear magnetic resonance data show how molecular chaperones recognize and prevent aggregation and misfolding of unfolded proteins. [Also see Perspective by Gamerdinger and Deuerling] Molecular chaperones prevent aggregation and misfolding of proteins, but scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms. We report the solution structure, dynamics, and energetics of three trigger factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. Nuclear magnetic resonance (NMR) relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion, but as the number and length of the PhoA regions engaged by TF increase, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and, by acting as unfoldases and holdases, prevent the aggregation and premature (mis)folding of unfolded proteins. Recognize and Protect Molecular chaperones play a key role in maintaining protein homeostasis in the cell by preventing protein aggregation and misfolding. Chaperone-substrate complexes tend to be large and dynamic, making structure determination challenging. Saio et al. (10.1126/science.1250494; see the Perspective by Gamerdinger and Deuerling) used advanced NMR spectroscopy techniques to determine the structure of three trigger factor (TF) chaperone molecules in complex with the unfolded substrate, alkaline phosphatase (PhoA), and of each of the TFs in complex with the relevant region of PhoA. TF binds at multiple sites on PhoA through hydrophobic contacts, thus shielding these residues from solvent and preventing aggregation. The stability of the complex increases as longer PhoA regions are engaged by TF, and the multivalent binding keeps the substrate in an extended conformation.

Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function

SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase. SecA comprises a dimerization C‐terminal domain fused to an ATPase N‐terminal domain containing conserved DEAD helicase motifs. We show that the N‐terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2. NBD1, a rigid nucleotide‐binding domain, contains the minimal ATPase catalytic machinery. IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controling ADP release and optimal ATP catalysis at NBD1. IRA2 is flexible and can undergo changes in its α‐helical content. The C‐terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function. Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP‐bound state and unnecessary ATP hydrolysis cycles are prevented. Two DEAD family motifs in IRA2 are essential for IRA2–NBD1 binding, optimal nucleotide turnover and polypeptide translocation. We propose that translocation ligands alleviate C‐terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1. DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.

Signal peptides are allosteric activators of the protein translocase

Extra-cytoplasmic polypeptides are usually synthesized as ‘preproteins’ carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, ‘triggering’ that drives the translocase to a lower activation energy state; second, ‘trapping’ that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, ‘secretion’ during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases.

Identification of the Preprotein Binding Domain of SecA

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these “specificity domains” is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.

Bacterial preprotein translocase: mechanism and conformational dynamics of a processive enzyme

Preprotein translocase, the membrane transporter for secretory proteins, is a processive enzyme. It comprises the membrane proteins SecYEG(DFYajC) and the peripheral ATPase SecA, which acts as a motor subunit. Translocase subunits form dynamic complexes in the lipid bilayer and build an aqueous conduit through which preprotein substrates are transported at the expense of energy. Preproteins bind to translocase and trigger cycles of ATP binding and hydrolysis that drive a transition of SecA between two distinct conformational states. These changes are transmitted to SecG and lead to inversion of its membrane topology. SecA conformational changes promote directed migration of the polymeric substrate through the translocase, in steps of 20–30 aminoacyl residues. Translocase dissociates from the substrate only after the whole preprotein chain length has been transported to the trans side of the membrane, where it is fully released.