1. Quaternary Dynamics of the SecA Motor Drive Translocase Catalysis
Giorgos Gouridis, Spyridoula Karamanou, Marios Frantzeskos Sardis, Martin Alexander Schärer, Guido Capitani, Anastassios Economou 
Molecular Cell 
Volume 52, Issue 5, Pages (December 2013)
DOI: /j.molcel
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2. Figure 1 Identification of the SecA Dimer Interface
(A) One ecSecA protomer (Papanikolau et al., 2007) modeled after mtSecA (Sharma et al., 2003). NBD: nucleotide binding domain; IRA2: intramolecular regulator of ATPase 2; SD: Scaffold domain, IRA1: intramolecular regulator of ATPase 1; WD: wing domain, PBD: preprotein binding domain.
(B) Two ecSecA dimers modeled after mtSecA 1NL3_1 (Sharma et al., 2003) and bs1M6N (Hunt et al., 2002; Sharma et al., 2003; Zimmer et al., 2006) (see also Figure S1). Fixed protomer (as in A): light green; moving one: cyan, with its IRA1 and SD domains (darker blue) acting as guides. The rotation angle to interconvert between these dimers is indicated. See also legend to Table S1.
(C) Summary of stabilizing noncovalent interactions in (B) dimers (below 5Å; presented in detail in Table S1). Lines are colored according to structures; width is proportional to the strength of the interaction. Interacting residues belong to the indicated SecA domains or substructures (α0; α1; SD; IRA1; WD) of the two protomers. See also Figure S1 and Movies S1.
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3. Figure 2 Equilibrium of SecA Conformers in Solution
(A) GPC-MALLS-QELS analysis of wild-type SecA (2.5 μM) and the monomeric SecA(Δα0/α1-6A), in 50 mM Tris pH:8; 50 mM KCl. The UV traces of the two chromatograms were superimposed, together with the measured mass (red circles) and hydrodynamic diameter (black circles).
(B) Superimposed UV traces from preparative GPC of wild-type SecA (20 μM) at the indicated KCl concentration. In the high salt chromatogram (red), monomeric SecA (M) and the residual salt-resistant dimer (SRD) are indicated. In the low salt chromatogram (black), dimeric SecA consists of SRD and electrostatic dimer (ED; i.e., the dimer that monomerizes by high salt; see C below).
(C and D): SecA conformers in solution: quaternary state (C) and hydrodynamic dimensions (D).
SecA was analyzed by two consecutive steps of preparative GPC. First in 1M KCl, to separate the SRD and M species (as in B; red line). The M species was then analyzed in 50 mM KCl to separate the ED (previously monomerizing in high ionic strength) from the Monomer (remaining monomeric even in low ionic strength). Areas under the chromatographic curves were calculated (Matlab), percentages were assigned as follows: 100% of any SecA = M + ED + SRD and are shown in (C). Peak fractions of preparative GPC were collected and analyzed on analytical GPC-MALLS-QELS to determine mass and hydrodynamic diameters (shown in D). Only the main species from each population is shown, except in the case of α1-6A, which has two main species. Conformer colors: ED (dark blue), SRD (light blue), Monomer (white).
(E) Activation energies (Ea) of SecA derivatives in solution (lanes 1–4) or in the presence of wild-type SecYEG-IMVs (lanes 5–8) or/and preprotein (lane 9), as indicated. The color of soluble proteins (lanes 1–4) indicates their conformer state as determined in (C) and (D). In lanes 5–9 (gray) direct measurements of mass/dimensions under these conditions are not available.
(F) Equilibrium diagram of SecA states (M, SRD, ED) in solution. Soluble SecA is predominantly ED (∼95%). The PrlD23 mutation stabilizes the triggered dimer (TD) in solution. High ionic strength favors the monomer. However, at cellular SecA concentrations this is prevented (Wowor et al., 2011). See also Figure S2 and Table S2.
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4. Figure 3
Membrane Binding and Catalytic Ability of Dimeric and Monomeric SecA
(A) Dissociation constants (KD) and binding stoichiometries of wild-type SecYEG (“SecY2”) to either wild-type dimeric SecA (“SecA2”) or monomeric SecA(Δα0/α1-6A) (“SecA1”) (see also Table S3).
(B) Dissociation constants (KD) and binding stoichiometries of the holoenzymes formed in (A) (as indicated), to proPhoA (see also Table S3).
(C) Left: In vivo genetic complementation, at 42°C, of a secAts E.coli strain (BL21.19) by the wild-type secA or the secA monomer, in the absence or presence of 10μM IPTG (as indicated). Right: In vivo proPhoA secretion was monitored under similar conditions. Error bars represent standard deviation values (n = 3).
(D) The amount of proPhoA translocated in vitro by the SecA monomer is linearly correlated with the amount of dimer it can form. The translocation efficiency (black circles) or the amount of dimer formed (white circles) by the monomer, at a given concentration, was expressed as a percentage of the corresponding wild-type dimeric activity (considered 100%) (see Supplemental Experimental Procedures).
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5. Figure 4 Functional Analysis of SecA and Derivatives at 37°C
(A) In vitro translocation of proPhoAcys- by the indicated SecA proteins, into wild-type (left panel) or PrlA4 (right panel) SecYEG-IMVs. Values were expressed as a percentage of the wild-type translocation efficiency (taken as 100%).
(B and C) The Kcat values of basal (B), membrane (M), and translocation (T) ATPase of the indicated SecA proteins were determined. Stimulation of membrane (panel B: M Kcat/B Kcat) or translocation (panel C: T Kcat/M Kcat) ATPase, using wild-type or SecY/PrlA4-EG-IMVs is shown (as indicated).
(D) Activation energies (Ea) of SecA derivatives under translocation conditions, using wild-type or SecY/PrlA4-EG-IMVs (as indicated).
In all panels, error bars represent standard deviation values (n > 3). See 4–40°C range in Figure S4.
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6. Figure 5 Membrane Insertion of SecA Proteins
(A) ATP binding drives a “membrane-inserted” conformation of SecA. Wild-type SecA and wild-type SecYEG-IMVs were incubated with nucleotide (as indicated). Lane 9: soluble SecA. The amount of the protease-protected SecA C domain (Economou and Wickner, 1994) (see also Supplemental Experimental Procedures) was expressed as a percentage of that measured in the presence of AMP-PNP (considered 100%).
(B) The triggered translocase stabilizes a specific SecA membrane-bound conformation. Wild-type SecA was bound to wild-type or SecY/PrlA4-EG-IMVs (as indicated).
(C) Only dimeric SecA becomes membrane-protected; triggered monomeric SecA cannot. SecA derivatives were bound to wild-type or SecY/PrlA4-EG-IMVs, in the presence of AMP-PNP (as indicated).
(D) SRD is an intermediate of ED, toward the TD state. ED, SRD, and TD purified SecA proteins were incubated with wild-type SecYEG-IMVs (as indicated). In all panels, error bars represent standard deviation values (n > 3).
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7. Figure 6 SecY:SecA Ratio Finely Tunes Distinct Steps of Translocation
(A) The Ea of the triggered wild-type translocase (black circles) and stimulation of membrane turnover (white circles) were determined as a function of SecA:SecY ratio.
(B) In vitro proPhoA translocation (black circles; see also Figure S6A), trapping of PhoA mature chain (arrowheads) (Gouridis et al., 2009) and stimulation of translocation turnover (white circles) were determined as a function of SecY:SecA ratio. Values were expressed as a percentage of the maximum value obtained for each function. Error bars in (A) and (B) represent standard deviation values (n > 3).
(C) Intracellular SecA and SecY concentrations were determined for the indicated E.coli strains, using quantitative Western blotting with α-SecY and α-SecA (see Supplemental Experimental Procedures) and the native SecY/SecA ratio was calculated (n = 3).
(D) In vivo genetic complementation of the secAts E.coli strain BL21.19 by the wild-type secA, in the presence of the indicated IPTG. Identical plates incubated at 30°C show full growth under all conditions (not shown).
(E) proPhoA secretion by the indicated SecA proteins (WT [black]; Δα0 [orange] α1-6A [blue], Δα0/α1-6A [magenta]) was determined in vivo (empty symbols) and in vitro (filled in symbols), as a function of SecA concentration (x axis). To directly compare the secretion efficiency of the different mutants, all values were expressed as a percentage of the wild-type SecA maximum secretion efficiency, obtained in vivo at 4.5 μM (no IPTG added) and in vitro at 0.2 μM. All the experimental points for one protein were combined and optimally fitted in a single curve (see also Supplemental Experimental Procedures). Preprotein secretion by the various SecA proteins as a function of their concentration is presented in the same graph for a direct comparison. Error bars (shown only for the wild-type and monomeric SecA) represent standard deviation values (n = 3).
(F) Yield (left) and time kinetics (right) of proPhoAcys- translocation into wild-type SecYEG-IMVs, in vitro. Left: SecA(Δα0/α1-6A)/PrlD23 retains 10% of the wild-type SecA translocation efficiency under the same experimental conditions (12 min, 37°C). Right: Irrespective of the yield difference, the accumulation of translocated proPhoAcys- exhibits similar kinetics in wild-type SecA or SecA(Δα0/α1-6A)/PrlD23. In this panel, translocation efficiency at 24 min for either SecA was considered 100%; all other values were expressed as a percentage of those. Experimental data were optimally fitted with lines. Error bars (shown only for the wild-type SecA) represent standard deviation values (n = 3). See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Structural Basis of SecY-Mediated Control of SecA Monomerization and Model of the Mechanism of Protein Translocation
(A) Left: Model of the E.coli translocase based on the T. maritima structure (Zimmer et al., 2008). Right: The ecSecA dimer (α, cyan; β, faint green), modeled after the mt1NL3_1 dimer (Figure 1B), was docked on SecYEG through structural alignment of SecAα.
(B) SecAα (left; surface representation; colored as in Figure 1A) rotated by ∼90° compared to (A), sterically clashes its second protomer (SecAβ; middle, light green) with SecY (right, yellow). Only structural elements of SecAβ (tubes; light green) or SecY (tubes; yellow; detailed in the Supplemental Information) that interact with SecAα (cyan) are shown.
(C) Model of stepwise process of protein translocation, emphasizing the roles of distinct quaternary states of the SecA motor (see text for details). SecYEG (white rectangles) and the SecA dimer (light blue and green circles) subunits are enlarged in steps III–VII to represent the triggered translocase, characterized by its low activation energy. Secretion: segmental export of the mature domain. Preprotein/SecA complexes of lower affinity can also form in the cytoplasm (not shown).
Molecular Cell  , DOI: ( /j.molcel )
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