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. 2007 Jun 20;26(12):2904-14.
doi: 10.1038/sj.emboj.7601721. Epub 2007 May 24.

Preprotein-controlled catalysis in the helicase motor of SecA

Spyridoula Karamanou  1 , Giorgos Gouridis, Efrosyni Papanikou, Giorgos Sianidis, Ioannis Gelis, Dimitra Keramisanou, Eleftheria Vrontou, Charalampos G Kalodimos, Anastassios Economou
Affiliations

Preprotein-controlled catalysis in the helicase motor of SecA

Spyridoula Karamanou et al. EMBO J. .

Abstract

The cornerstone of the functionality of almost all motor proteins is the regulation of their activity by binding interactions with their respective substrates. In most cases, the underlying mechanism of this regulation remains unknown. Here, we reveal a novel mechanism used by secretory preproteins to control the catalytic cycle of the helicase 'DEAD' motor of SecA, the preprotein translocase ATPase. The central feature of this mechanism is a highly conserved salt-bridge, Gate1, that controls the opening/closure of the nucleotide cleft. Gate1 regulates the propagation of binding signal generated at the Preprotein Binding Domain to the nucleotide cleft, thus allowing the physical coupling of preprotein binding and release to the ATPase cycle. This relay mechanism is at play only after SecA has been previously 'primed' by binding to SecYEG, the transmembrane protein-conducting channel. The Gate1-controlled relay mechanism is essential for protein translocase catalysis and may be common in helicase motors.

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Figures

Figure 1
Figure 1
(A) Ribbon representation of ecSecA (Papanikolau et al, 2007). NBD, nucleotide binding domain (blue); IRA2, intramolecular regulator of the ATPase 2 (light blue); PBD, preprotein binding domain (magenta), C-domain (green), ATP (red); SD, scaffold domain. (B) The Kcat values (pmoles Pi/pmol SecA protomer/min) of basal, membrane (urea-treated IMVs; 17 μg protein per milliliter) and translocation (IMVs plus 60 μg/ml proOmpA) ATPase activities of SecA were determined as a function of temperature. The peak Kcat value of basal ATPase was taken as 100% and all other values were expressed as its percentage. Averaged data (n=8) of SecA basal ATPase were used in subsequent graphs. (C) Basal ATPase activities of SecA, SecA(D649A), SecA(W775A) (as in panel B). The peak Kcat value for each ATPase (Supplementary Table 1) was taken as 100% and all other values were expressed as its percentage. (D) Basal ATPase activity of SecAΔC (as in B) in the presence or absence of 10X molar excess of C34 (as indicated). The peak Kcat value for the SecAΔC ATPase (Supplementary Table 1) was taken as 100% and all other values (± C34) were expressed as its percentage. Averaged data (n=12) of SecAΔC were used in subsequent graphs.
Figure 2
Figure 2
(A) PBD deletions, point mutations (red crosses) and secondary structure are indicated on a region map. β1 and β7 (pink) together form the antiparallel sheet of the Stem. Between them lie the two globular halves of the Bulb (1, magenta and 2, purple). See also Figure 1A. (B) Basal ATPase activities of SecAΔC, SecAΔC(R347A), SecAΔC(W349A) and SecAΔC(L319P) (as in Figure 1D). (C) Chemical shift change (Δδ) as a function of temperature mapped onto the structure of PBD. Higher Δδ values indicate temperature-induced unfolding. (D) SecA or SecAΔC (245 pmol in 100 μl buffer B) were treated (13 min) by limited trypsinolysis at 4°C (0.25 μg/ml) or 37°C (0.025 μg/ml). Trypsin was inactivated with Pefabloc (9 mM). Following SDS–PAGE, polypeptides were immunostained by α-PBD-specific antibody and were identified by N-terminal sequencing.
Figure 3
Figure 3
Basal thermal ATPase activities of SecAΔC (A) and SecA (B) in the presence or absence of pCH5EE (‘preprotein'; Baud et al, 2005; Papanikou et al, 2005) (as in Figure 1C).
Figure 4
Figure 4
(A) The Gate1 region of ecSecA (Papanikolau et al, 2007) and the corresponding regions in Vasa (Sengoku et al, 2006) and UvrB (Theis et al, 1999). Motif II residue side chains are colored in red. (B) Basal ATPase activities of SecAΔC(R566A), SecAΔC(D217A) and SecAΔC (as in Figure 1D). SecAΔC basal ATPase activity was also determined in the presence of 0.55 M KCl (as indicated). (C) Basal ATPase activities of SecAΔC, SecAΔC(ΔBulb) and SecAΔC(ΔBulb/R566A) (as in Figure 1D). The cartoons (inset) are schematic representations of the three proteins; red cross indicates R566A mutation. (D) Equilibrium dissociation constants (Kd) of SecAΔC and derivatives for the fluorescent analog MANT-ADP plotted as a function of temperature were determined as described (Vrontou et al, 2004). (E) Observed enthalpy change (ΔH) as a function of temperature for the SecAΔC and SecAΔC(R566A) interaction with MgADP (in bufer B with 100 mM KCl). Data were determined using ITC and analyzed as described (Keramisanou et al, 2006).
Figure 5
Figure 5
(A) SecAΔC and derivatives (0.9 mg/ml in buffer B) were treated with trypsin (25 μg/ml; 4°C; 13 min) in the absence or presence of ADP (1mM) (as indicated) or a three-fold molar excess of C34 (wherever indicated). Following SDS–PAGE, polypeptides were immunostained by α-IRA2-specific antibody. (B) Basal ATPase activities of SecA, SecA(R566A) and SecA(R347A) (as in Figure 1C). (C) Equilibrium dissociation constants (Kd) of SecA and SecA(R566A) for MANT-ADP plotted as a function of temperature. Two curves (dotted lines) from Figure 4D are included for comparison.
Figure 6
Figure 6
(A) In vivo genetic complementation test of the BL21.19 secAts strain at 42°C (Mitchell and Oliver, 1993) using SecA Gate1 mutants. BL21.19 cultures with pET5 vector alone or its derivatives with cloned secA or secA(R566A) or secA(D217A) or secA(D212A), secA(E397A) genes, grown at 30°C were adjusted to the same density. The indicated dilutions were spotted on LB/ampicillin plates and incubated (20 h; 42°C). (B) Interactions at Gate1 detected by crystallography. NBD residues involved in bonding with R566 of IRA2 in the M. tuberculosis SecA (Sharma et al, 2003). (C) In vitro protein translocation of SecA ‘Gate1' derivatives. ATP-driven in vitro translocation of proOmpA-His (0.1 mg/ml) into SecYEG proteoliposomes (20 μg protein/ml; 100 μl reactions in buffer B, BSA (0.5 mg/ml), 1 mM ATP, 1 mM DTT and SecB (0.4 mg/ml)) catalyzed by SecA or the indicated derivatives. Lane 1: 10% of undigested proOmpA-His input, lane 4: membranes were dissolved with Triton X-100 (1%) before proteinase K (1 mg/ml) addition. Proteins were TCA precipitated (15%), analyzed by SDS–PAGE and immunostained with α-His antibody. (D) Basal, membrane and translocation ATPase activities of SecA(R566A) (as in Figure 1B).
Figure 7
Figure 7
Hypothetical model of preprotein-controlled PBD–Gate1–IRA2 conformational relay that leads to activation of the SecA DEAD motor ATPase (see ‘Discussion' for details). Irregular contours denote conformational changes. Red star, ADP; orange line, preprotein.
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References

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