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. 2001 Mar 1;20(5):961-70.
doi: 10.1093/emboj/20.5.961.

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

G Sianidis  1 , S Karamanou, E Vrontou, K Boulias, K Repanas, N Kyrpides, A S Politou, A Economou
Affiliations

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

G Sianidis et al. EMBO J. .

Abstract

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 controlling ADP release and optimal ATP catalysis at NBD1. IRA2 is flexible and can undergo changes in its alpha-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.

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Figures

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Fig. 1. Map of SecA and truncated derivatives. SecA consensus motifs (60% identity or similarity) were determined as described (Bailey and Gribskov, 1999) and by visual inspection. The corresponding DEAD superfamily II motifs are indicated. Family motifs (dark boxes) in E.coli SecA are: motif I (aa 66–82; EAFAVVREA[S/A][R/K]RVLGXR); motif II (aa 83–139; [P/H][F/Y]DVQLIGG[M/I][V/A]LHXGXIAEM[K/R]TGEGKTL[V/T]ATL[P/A][A/V]YLNAL[S/T]GKGVHVVTVNDYLA[R/K]RD); motif III (aa 149–156; FLGL[T/S]VG[V/L]); motif IV (aa 166–191; [R/K][R/K]XAYX[A/C]DITY[A/G]TN[N/S]E[F/L]GFDYLRDN[M/L]); motif V (aa 205–227; [F/Y]AIVDEVDSILIDEARTPLIISG); motif VI (aa 333–361; V[V/L]IVDEFTGR[V/L][M/L]XGRR[Y/W]S[D/E]GLHQAIEAKE); motif VII (aa 371–397; TLA[T/S]IT[Y/F]QN[Y/F]FR[L/M]YXKL[A/S]GMTGTAXTE); motif VIII (aa 404–430; IYX[L/M]XV[V/I]XIPTNRP[M/V]XRXDXXDL[I/V]YX[T/S]); motif IX (aa 449–466; GQP[V/L]LVGT[I/T]S[V/I]EXSE[L/Y]LS); motif X (aa 494–516; IXHXVLNAKXXX[R/K]EAXI[I/V]AXAGXXGAVTIATNMAGRG TDIXLG); motif XI (aa 532–599; GGLX[I/V]IGTERHESRRID NQLRGR[A/S]GRQGD[P/A]GXSRFYLSLEDXL[M/L]R[I/L]F[A/G]); motif XII (aa 630–663; AQ[K/R]KVE[G/A]XXN[F/Y][D/E][W/L]RKQLXXYDDV[M/L]XXQRXXIYXXR); motif XIII (aa 767–818; [L/I]LXXIDXXWREHLXXMDXLRXGIXLR[G/A]YAQKDPLXE YXXE[G/A][Y/F]X[L/M]FXXM[L/M]XX[I/L]). Symbols used: X = any residue; alternative substitutions for the same residue are shown in brackets.
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Fig. 2. ADP release from SecA is rate limiting for multiple rounds of ATP hydrolysis. (A) Single versus multiple rounds of SecA ATP hydrolysis. SecA or N68 (10 pmol) or no protein (10 µl reactions) was incubated at the indicated temperature with [γ-32P]ATP (1 µM, 1:1 molar ratio, 1 min; or 1 mM, 1:1000, 10 min). Hydrolysis was stopped by proteinase K (5 µl of 10 mg/ml stock) at 4°C and samples were analysed by TLC and autoradiography. AMP and ADP markers are indicated. (B) Effect of temperature on SecA ATP hydrolysis. SecA or N68 (50 pmol in 10 µl) was incubated with 1 mM [γ-32P]ATP (37 or 4°C, 5 min). At the indicated time point, proteinase K was added and samples were analysed by TLC (as in A) and quantitated (see Materials and methods). (C) ADP release is rate limiting for multiple ATP turnovers by SecA. SecA or N68 (100 pmol) or no protein (20 µl reactions) was incubated with 100 µM [α-32P]ATP (10 min, 4°C). One-third of the reaction was treated with proteinase K (4°C; control), one-third was subjected to centrifugal gel filtration (4°C; CGF) and one-third was incubated at 37°C (5 min) before CGF (as indicated). Samples were analysed as in (A). (D) ADP causes an increase in the melting temperatures of SecA and N68. Thermal denaturation curves, in the presence or absence of ADP (2 mM), were obtained by monitoring ellipticity at 222 nm by far-UV CD, while heating the protein samples (200 µl, 200 µg/ml in 5 mM MOPS buffer pH 7.5, 5 mM MgCl2) at 50°C/h and were analysed as described (Karamanou et al., 1999).
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Fig. 3. NBD1 is the minimal SecA ATPase domain and is activated by IRA2. (A) IRA2 stimulates NBD1 ATP hydrolysis. N1–479 or N1–479D209N (100 pmol in 10 µl) supplemented with IRA2 (indicated molar ratios) was incubated with 0.5 mM [γ-32P]ATP (15 min; 37°C). Hydrolysis was determined as in Figure 2B. (B) IRA2 activates NBD1 ATPase through direct physical interactions. Reactions as in (A) (1:1 molar ratio shown), with BSA used as carrier, were analysed by native PAGE (4–20% gradient) and Coomassie Blue staining. An asterisk represents reconstituted complex. IRA2 migrates on native gels as a fuzzy, weakly staining band. (C) NBD1 physical association with IRA2. N1–479, N1–263 or control proteins (250 nM in 100 µl of buffer B) were added to biosensor-immobilized IRA2 on an amino surface. IRA2 (250 nM; buffer B) or control proteins were bound to immobilized N1–263. BSA, bovine serum albumin; GDH, glutamate dehydrogenase.
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Fig. 4. IRA2 is required for nucleotide binding and hydrolysis at NBD1. (A) Basal ATPase activity of SecA and IRA2 mutant derivatives determined by the malachite green method (as in Lill et al., 1990). (B) IRA2 mutations affect NBD1–IRA2 binding. IRA2 mutants (500 nM; buffer B, 100 mM NaCl, 5% glycerol) were added to biosensor-immobilized N1–479. The binding response is expressed in millidegrees. (C) ATP hydrolysis kinetics of SecA, N68 and G510A and R566A mutant derivatives (50 pmol protein in 10 µl reactions) at 4°C (1 mM [γ-32P]ATP; analysed as in Figure 2B). (D) Nucleotide occupancy of SecA IRA2 mutants. Samples (50 pmol protein in 10 µl reactions) prior to (lanes 2–7) or after CGF (lanes 8–13) were analysed as in Figure 2C. (E) ATP pre-steady-state kinetics of SecA and N68, R509K and R577K mutant derivatives (50 pmol protein, 5 µM [α-32P]ATP in 10 µl reactions analysed as in Figure 2B). Hydrolysis is expressed as a percentage of that of equimolar amounts of wild-type SecA or N68, under identical conditions.
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Fig. 5. The C-terminal domain binds to NBD1 and IRA2. (A) Basal ATPase activity of SecA and mutant derivatives (as in Figure 4A). (B) Binding of N68 and its IRA2 mutant derivatives to biosensor-immobilized C34 (as in Figure 4B). (C) The indicated purified domains (ligate) were assayed for binding to biosensor-immobilized C34, NBD1 or IRA2 (as in Figure 4B). GDH, glutamate dehydrogenase.
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Fig. 6. The IRA2 domain is essential for protein translocation and life. (A) IRA2 mutant in vivo test. BL21.19 cultures carrying pET5 vector alone or its derivatives with cloned secA, secAR509K, secAG510A, secAR566A or secAR577K genes were adjusted to the same density. Dilutions (10n) were spotted on LB/ampicillin plates and incubated at 42°C. (B) In vitro preprotein translocation in SecYEG-proteoliposomes of SecA IRA2 mutants (as in Karamanou et al., 1999). Lane 1: 25% of input [35S]proOmpA. Triton X-100 (1% v/v) was added prior to trypsin digestion (lanes 2–12). (C) Basal, membrane and translocation ATPase activities of the IRA2 SecA mutant proteins (determined as in Lill et al., 1990).
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Fig. 7. Domain organization model of the SecA protomer (see text for details).
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References

    1. Ali J.A. and Lohman,T.M. (1997) Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science, 275, 377–380. - PubMed
    1. Ausubel F.M., Brent,R., Kingston,R.E., Moore,D.D., Smith,J.A., Seidman,J.G. and Struhl,K. (1994) Current Protocols in Molecular Biology. John Wiley and Sons, NY.
    1. Bailey T.L. and Gribskov,M. (1999) Combining evidence using P-values: application to sequence homology searches. Bioinformatics, 14, 48–54. - PubMed
    1. Bianco P.R. and Kowalczykowski,S.C. (2000) Translocation step size and mechanism of the RecBC DNA helicase. Nature, 405, 368–372. - PubMed
    1. Breukink E., Nouwen,N., van Raalte,A., Mizushima,S., Tommassen,J. and de Kruijff,B. (1995) The C terminus of SecA is involved in both lipid binding and SecB binding. J. Biol. Chem., 270, 7902–7907. - PubMed

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