Portal de Periódicos da CAPES

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

2001; Springer Nature; Volume: 20; Issue: 5 Linguagem: Inglês

10.1093/emboj/20.5.961

1460-2075

Giorgos Sianidis,

RNA Research and Splicing

Article1 March 2001free access Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function Georgios Sianidis Georgios Sianidis Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Spyridoula Karamanou Spyridoula Karamanou Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Eleftheria Vrontou Eleftheria Vrontou Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Kostantinos Boulias Kostantinos Boulias Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Kostantinos Repanas Kostantinos Repanas Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Nikos Kyrpides Nikos Kyrpides Department of Basic Sciences, Medical School, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Anastasia S. Politou Anastasia S. Politou Integrated Genomics, Inc., 2201 West Campbell Park Drive, Chicago, IL, 60612 USA Search for more papers by this author Anastassios Economou Corresponding Author Anastassios Economou Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Georgios Sianidis Georgios Sianidis Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Spyridoula Karamanou Spyridoula Karamanou Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Eleftheria Vrontou Eleftheria Vrontou Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Kostantinos Boulias Kostantinos Boulias Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Kostantinos Repanas Kostantinos Repanas Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Nikos Kyrpides Nikos Kyrpides Department of Basic Sciences, Medical School, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Anastasia S. Politou Anastasia S. Politou Integrated Genomics, Inc., 2201 West Campbell Park Drive, Chicago, IL, 60612 USA Search for more papers by this author Anastassios Economou Corresponding Author Anastassios Economou Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece Search for more papers by this author Author Information Georgios Sianidis1, Spyridoula Karamanou1, Eleftheria Vrontou1, Kostantinos Boulias1, Kostantinos Repanas1, Nikos Kyrpides2, Anastasia S. Politou3 and Anastassios Economou 1 1Institute of Molecular Biology and Biotechnology and Department of Biology, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece 2Department of Basic Sciences, Medical School, University of Crete, PO Box 1527, GR-711 10 Iraklio, Crete, Greece 3Integrated Genomics, Inc., 2201 West Campbell Park Drive, Chicago, IL, 60612 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:961-970https://doi.org/10.1093/emboj/20.5.961 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction One third of the cellular proteome is extracytoplasmic (Gerstein et al., 2000). Most secretory and membrane proteins are synthesized as preproteins that are delivered to membranes by chaperones and/or the signal recognition particle (Danese and Silhavy, 1998; Johnson and van Waes, 1999). Bacterial translocase comprises the integral membrane proteins SecYEGDFYajC and the membrane-associated ATPase motor SecA (Economou, 1998, 2000; Manting and Driessen, 2000). SecYEA comprise the essential enzyme core. Preproteins trigger ATP-driven cycling of SecA between a 'membrane-inserted' and a 'deinserted' state (Economou and Wickner, 1994). Powered by SecA, the translocase moves processively along the polymeric substrate (Economou, 1998) in defined steps (Schiebel et al., 1991; van der Wolk et al., 1997). The precise mechanisms of SecA ATP hydrolysis and the coupling of energy to protein translocation remain elusive. Secretory preproteins cross the membrane through a SecY–SecA channel (Joly and Wickner, 1993; Matsumoto et al., 1997) that may resemble a 'pore' (Meyer et al., 1999; Manting et al., 2000). SecA comprises two domains: the 68 kDa N-terminal domain and the 34 kDa dimerization C-terminal domain (Figure 1; Economou, 1998; Karamanou et al., 1999) that have been synthesized as independent polypeptides (termed N68 and C34, respectively). N68 and C34 bind to each other and reconstitute SecA (Karamanou et al., 1999; Dapic and Oliver, 2000; Nakatogawa et al., 2000). In cytoplasmic SecA, the N-terminal domain ATPase is repressed by the C-terminal domain IRA1 switch (previously IRA; Figure 1; Karamanou et al., 1999) by means of physical association. Figure 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. Download figure Download PowerPoint SecA is an ATP-dependent RNA helicase (Park et al., 1997) and contains conserved DEAD protein family motifs (Figure 1; Koonin and Gorbalenya, 1992). The collective term 'DEAD' represents five superfamilies of ATPases including bona fide nucleic acid helicases (de la Cruz et al., 1999; Hall and Matson, 1999). Walker box sequences A (Figure 1, motif II; Walker et al., 1982) and B (motif V) form a high-affinity nucleotide-binding site (NBD1) essential for SecA function (Matsuyama et al., 1990; Mitchell and Oliver, 1993; Economou et al., 1995). The region encompassing residues 500–650 (termed 'NBD2') was proposed to be responsible for a second, low-affinity nucleotide-binding activity (Mitchell and Oliver, 1993). This region is important for ATP hydrolysis (Karamanou et al., 1999) and protein translocation (Mitchell and Oliver, 1993; Economou et al., 1995). However, its function has remained unresolved since nucleotide binding to 'NBD2' has not been demonstrated experimentally; the proposed Walker boxes are non-canonical, with box B located outside the limits of the ATPase N-terminal domain (Karamanou et al., 1999). We now demonstrate that this region is not an independent nucleotide-binding or hydrolysis site. Instead, it is a regulatory domain that harbours two conserved DEAD family motifs. This novel intramolecular regulator of ATP hydrolysis (IRA2) is highly flexible and acts through binding to NBD1. IRA2 activates ATP hydrolysis and multiple nucleotide turnovers at NBD1 in trans and is essential for polypeptide translocation. Results ADP release is rate limiting for multiple ATP turnovers by SecA To study the mechanism of ATP hydrolysis by SecA, we employed thin-layer chromatography (TLC). Full-length SecA or the isolated N68 domain completely hydrolyse stoichiometric amounts of ATP at either 37 or 4°C, within 1 min (Figure 2A, lanes 1–6). In the presence of excess ATP, both enzymes catalyse multiple rounds at 37°C (Figure 2A, lanes 7–9, and B), but SecA is less efficient due to IRA1 suppression (Figure 2B; Karamanou et al., 1999). In contrast, at 4°C, only limited hydrolysis occurs (Figure 2A, lanes 10–12, and B). Since the ATP hydrolysis chemistry per se is not affected by low temperature (Figure 2A, lanes 1–6), there must exist a rate-limiting step downstream in the reaction cycle that represses multiple rounds of ATP hydrolysis at 4°C (Figure 2B). Figure 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). Download figure Download PowerPoint To determine this step, we investigated the nucleotide occupancy of SecA and N68 after ATP hydrolysis at 4°C by centrifugal gel filtration (CGF; Figure 2C). Strikingly, both enzymes are isolated in the excluded volume in an exclusively ADP-bound state (lanes 5 and 6), although 40–60% of the total nucleotide pool is ATP (lanes 2 and 3). Following CGF, most of the generated ADP remains bound to SecA and N68 (97 and 71%, respectively). A brief incubation at 37°C prior to CGF drastically reduces the amount of ADP co-isolated with N68 (only 2% remains bound; lane 9). This effect of elevated temperature is less dramatic for SecA (62% remains bound; lane 8), presumably due to the presence of the IRA1 switch (Karamanou et al., 1999). It appears that, following ATP hydrolysis at 4°C, ADP release from the N-terminal domain of SecA imposes a rate-limiting step for ATP turnover. At 37°C, the apparent ADP release rate, slow for SecA and fast for N68, could underly the observed 8-fold reduced ATP turnover of SecA compared with N68 (Table I). Table 1. Kinetic parameters of SecA and mutant derivatives Protein kcat (per min) SecA 4.6 N68(N1–610) 36.4 N1–263 0.1 N1–479 0.06 N1–547 0.08 IRA2(N462–610) n.d. N270–610 n.d. Class I SecAG510A 42.2 N68G510A 7.8 SecAR566A 19.6 N68R566A 12.3 Class II SecAR509K 4.5 N68R509K 2.1 SecAR577K 2.6 N68R577K 0.14 Enzymes were incubated (5 min; 37°C; buffer B) at a range of [γ-32P]ATP concentrations (30–2000 μM). Hydrolysis was determined as in Figure 1B. n.d., not detectable, i.e. following subtraction of the ATP background, values were zero or negative. We further investigated ADP retention by SecA or N68 using far-UV circular dichroism (CD). Thermal denaturation curves of the two enzymes reveal at least two discernible transitions centred at 39 and 48°C (Figure 2D). The more stable transition derives from a domain present in both SecA and N68. Interestingly, its apparent Tm increases dramatically by 15°C in the presence of ADP (Figure 2D; Table II). The lower transition is composite, could not be attributed to a single domain and was not analysed further. Clearly, ADP binds to the N-terminal domain and thermally stabilizes SecA. Table 2. Apparent melting temperatures (Tm) of SecA and mutant derivatives (as in Figure 1D and Karamanou et al., 1999) Protein Tm app −ADP +ADP SecA 48.9 62.5 N68(N1–610) 48.5 63.0 N1–263 49.1 57.0 N1–479 47.6 52.5 N1–547 48.2 56.1 IRA2(N462–610) 43.0 44.0 N270–610 43.5 44.0 Class I SecAG510A 41.9 48.0 SecAR566A 45.4 50.6 Class II SecAR509K 48.1 64.0 SecAR577K 47.9 65.1 NBD1 is the minimal catalytic machinery in SecA To delineate the N68 subdomains responsible for ADP binding and release, we aligned 68 SecA sequences. SecA contains 11 conserved motifs in the N-terminal domain (I–XI) and two in the C-terminal domain (XII and XIII; Figure 1; Karamanou et al., 1999). SecA belongs to DEAD superfamily 2 (Koonin and Gorbalenya, 1992; de la Cruz et al., 1999) and shows extensive similarity to DExH DNA repair and recombination enzymes (N.Kyrpides and A.Economou, unpublished results). We designed appropriately truncated N68 derivatives leaving conserved regions intact. The smallest fragment we generated, N1–263, fully encompasses the sequences comprising the high-affinity nucleotide-binding site NBD1 (Figure 1; Matsuyama et al., 1990; Mitchell and Oliver, 1993; den Blaauwen et al., 1999). N68 truncated derivatives were fused to oligohistidinyl tags and purified by metal affinity chromatography. All recombinant polypeptides are soluble, and their CD spectra verified that they are folded and stable (Table II and data not shown). Thermal denaturation curves demonstrated that all NBD1-containing polypeptides (N1–263, N1–479 and N1–547; Figure 1) retain the stable 48°C transition observed for N68 (Table II). This transition can therefore be attributed to NBD1. The apparent Tm of these polypeptides is significantly increased by ADP, albeit to a lower extent than that of SecA or N68 (5–8°C; Table II). In contrast, ADP does not cause any appreciable increase in the apparent Tm of the N270–610 or N462–610 fragments (Figure 1; Table II). We next tested the truncated N68 derivatives for the subsequent reaction step, ATP hydrolysis. N1–263, N1–479 and N1–547 can hydrolyse ATP, although very inefficiently compared with SecA or N68 (Table I). This low ATPase activity results from catalysis at NBD1 since it is eliminated by introduction of the D209N mutation (see below and data not shown) that abolishes NBD1 ATP hydrolysis in both SecA and N68 (Mitchell and Oliver, 1993; Economou et al., 1995). Fragments devoid of NBD1 (N270–610 or N462–610) display no detectable ATPase activity (Table I). We conclude that N1–263 fully encompasses the minimal nucleotide-binding and hydrolysis machinery in SecA. Nevertheless, residues on N462–610 seem to be required for both full-scale ADP-driven stabilization of SecA and high ATP turnover. We demonstrate below that this region, hereafter termed IRA2, is an essential intramolecular regulator of ATP hydrolysis at NBD1. IRA2 can stimulate ATP hydrolysis at NBD1 The deletion analysis presented above suggested that the N-terminal domain of SecA may comprise distinct catalytic and regulatory elements. We directly tested this hypothesis in vitro by supplying N1–479 with purified IRA2, the region missing from N1–479 but present on N68 (Figure 1). This led to a remarkable 15-fold activation of N1–479 ATPase activity, at 37°C (Figure 3A) but not at 4°C (data not shown). Analysis of these reactions by native PAGE reveals formation of N1–479–IRA2 complexes, visible as a species migrating more slowly than N1–479 or IRA2 alone (Figure 3B, lanes 1–3). In the same assay, N1–479D209N cannot become activated (Figure 3A) although it binds IRA2 as efficiently as the wild-type N1–479 (Figure 3B, compare lanes 5 and 3). This indicates that the stimulated ATP hydrolysis takes place at NBD1 and does not result from activation of a cryptic IRA2 ATPase activity. Figure 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. Download figure Download PowerPoint To quantitate IRA2 binding and to define the N1–479 region responsible for the association, we used an optical biosensor (Figure 3C). N1–263 (lane 2) and N1–479 (lane 1) bind efficiently to immobilized IRA2, while control proteins do not (lanes 3 and 4). Similarly, in the reverse experiment, IRA2 binds efficiently to immobilized N1–263 (compare lanes 5 and 2). We conclude that IRA2 binds specifically to NBD1 and stimulates its ATPase activity. Temperature affects the secondary structure of the IRA2 domain Low temperature prevents ADP release from the N-terminal domain of SecA and, therefore, ATP turnover, while elevated temperature has the reverse effect (Figure 2B and C; Schmidt et al., 2000). How is temperature 'sensed' by N68 and SecA? To address this, we examined the secondary structure of SecA, N68, N1–263, N1–479 and IRA2 at various temperatures, by far-UV CD. All these polypeptides are predominantly α-helical at 4°C (42–46%; data not shown). Helicity values were also determined at higher temperatures and expressed as a percentage of values obtained at 4°C (taken as a 100% baseline; Table III). Significantly, the α-helicity in IRA2 decreases upon temperature increase, suggesting extensive structural flexibilty. In contrast, the helicity of N1–263 or N1–479, which are devoid of IRA2, is not affected significantly in the temperature range examined. Interestingly, the helical content of both SecA and N68 is significantly decreased within the same temperature range, suggesting that IRA2 may be the main element contributing to partial endothermal melting of SecA or N68. Table 3. Temperature-induced α-helicity changes Polypeptide Relative α-helicity (%) 4°C 15°C 24°C 37°C SecA 100 99 97 49 N68(N1–610) 100 96 95 52 N1–263 100 95 92 75 N1–479 100 100 99 92 IRA2(N462–610) 100 92 87 38 Ellipticity at 222 nm is directly related to α-helical content and was derived from far-UV CD scans as described (Karamanou et al., 1999). Helicity at 4°C is expressed as 100%. Values at higher temperatures were normalized accordingly. Mutations in the DEAD motifs of IRA2 affect ATP turnover at NBD1 To elucidate further the molecular mechanism of IRA2 function, we sought to isolate point mutants with altered properties. SecA proteins contain only two conserved motifs (X and XI; DEAD family motifs V and VI; Figure 1) within the IRA2 domain. We generated point mutations in 100% conserved residues within SecA motifs X and XI and examined them together with the R509K mutant isolated previously (Mitchell and Oliver, 1993; Economou et al., 1995). Mutations, originally generated on His-SecA, were also transferred to N68 and IRA2 constructs. None of the mutations affects SecA or N68 secondary structure and folding (Table II and data not shown). Of 16 mutations, three had minor phenotypic changes and were not studied further. The remaining mutants had strong phenotypes and fell sharply into two classes. Since mutations in motifs X and XI result in similar phenotypes, we treated these motifs as a functional unit and focused on one representative mutant from each motif: class I (G510A, motif X; R566A, motif XI) and class II (R509K, motif X; R577K, motif XI). Class I mutations stimulate the basal ATPase of SecA (Figure 4A, lanes 1–3), while class II mutations slightly reduce it (lanes 4 and 5). The elevated basal SecA ATPase of class I mutants takes place at NBD1 since the D209N mutation abolishes it (compare lanes 2 and 3 with 7 and 8). Interestingly, both class I and class II IRA2 mutations reduce the ATPase of N68 (Table I), indicating that the presence of C34 in SecA may suppress the primary defect of class I IRA2 mutations (see below). Since IRA2 point mutations affect ATP turnover at NBD1 in either SecA or N68 (Figure 4A; Table I), we sought to identify specific elements of the NBD1 ATPase cycle that are under IRA2 control. Figure 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. Download figure Download PowerPoint IRA2 up-regulates ATP turnover at NBD1 by controlling ADP release First, we examined the effect of IRA2 mutations on the ability of SecA to interact with ADP. The ADP-induced thermal stabilization of SecA is not affected by the R509K and R577K substitutions (Table II). In contrast, SecAG510A and SecAR566A undergo limited ADP-driven stabilization (5–6°C; Table II), analogous to that seen with N-terminal domain truncations devoid of IRA2 (e.g. N1–263; Table II). An increased concentration of ADP (>10 mM) fails to enhance the thermal stability of class I mutants further (data not shown), suggesting that the observed defect does not result from reduced affinity of NBD1 for ADP. In full agreement with deletion analysis, these results firmly establish that although IRA2 is not required for nucleotide binding to NBD1, it is essential for full-scale ADP-driven stabilization of N68 and SecA (Table II). IRA2 associates with NBD1 (Figure 3B and C). To test whether class I mutations affect this property, we used an optical biosensor (Figure 4B). IRA2G510A and IRA2R566A exhibit decreased binding to immobilized N1–479 (compare lanes 2 and 3 with lane 1), while binding of IRA2 class II mutants is unaffected (compare lanes 4 and 5 with lane 1). Clearly, firm IRA2–NBD1 association is essential for full-scale ADP-mediated stabilization of SecA. ADP binding is a property inherent to NBD1 but not to IRA2 (see above and Table II). Up-regulation of SecA ATPase by class I IRA2 mutations (Figure 4A) could result from enhanced ADP release rates, due to the altered NBD1–IRA2 interaction described above (Figure 4B). If this was the case, class I mutants would be expected to bypass the suppression of ATP turnover seen in SecA and N68 under low temperature regimes (Figure 2B). This hypothesis was tested directly. Strikingly, G510A and R566A substitutions 'allow' both N68 and SecA to carry out multiple rounds of ATP hydrolysis at 4°C (Figure 4C). In full agreement with these results, the amount of ADP retained by SecAG510A and SecAR566A in CGF experiments at 4°C is drastically reduced (Figure 4D, compare lanes 9 and 10 with lane 8). R509K and R577K substitutions do not alter the ability of SecA to retain ADP (compare lanes 11 and 12 with lane 8). We conclude that IRA2 stimulates ATP turnover at NBD1 by controlling ADP release. The inability of IRA2 to release ADP from NBD1 (e.g. at 4°C or by loosened contact with NBD1) leads to repression of ATP turnover. Once ADP is released from NBD1, N68 and SecA can proceed to the next cycle of ATP hydrolysis. IRA2 optimizes the rate of catalysis at NBD1 Study of class II mutants reveals additional properties of IRA2. The ability of SecA to retain ADP (Figure 4D) and to become thermally stabilized (Table II) is not affected by class II mutants. Despite the fact that class II mutant IRA2 domains associate with NBD1 (Figure 4B), they fail to stimulate the N1–479 ATPase (data not shown). Moreover, the R509K mutation reduces ATP turnover in N68, and the R577K substitution down-regulates ATP turnover in SecA and drastically reduces it by 250-fold in N68 (Table I). It is striking that the effect of a single point mutation (R577K) in motif XI of IRA2 is as deleterious to NBD1 turnover as deletion of the whole motif XI (i.e. N1–547) or even deletion of the whoIe IRA2 domain (i.e. N1–263 or N1–479; Figure 1; Table I). These data indicate that in addition to controlling ADP release from NBD1, IRA2 may somehow be required for optimal catalysis. To elucidate this possibility further, we examined the effect of class II IRA2 mutations on NBD1 ATP hydrolysis by pre-steady-state kinetics (Figure 4E). This was possible since the nucleotide affinity at NBD1 is not altered by class II mutations (Km values; Mitchell and Oliver, 1993 and data not shown). Catalysis of stoichiometric amounts of ATP by SecA or N68 is complete by 1 min at either 4 or 37°C (Figure 2A). To facilitate comparison, the ATPase activity of class II mutant derivatives is expressed as a percentage of that derived from equimolar amounts of wild-type SecA or N68, under identical conditions. In agreement with steady-state kinetics (Table I), the R509K mutation does not affect catalysis in SecA but severely reduces it in N68 (Figure 4E). Catalysis of either SecA or N68 is severely affected by the R577K mutation, while low temperature exaggerates this effect (Figure 4E). These results suggest that the apparent 'loss of function' of class II IRA2 mutations is due to defective ATP hydrolysis at NBD1. We therefore propose that IRA2 has a dual role: it optimizes the rate of NBD1 catalysis and controls ADP release from NBD1. C34 contribution to NBD1–IRA2 regulation NBD1 is the minimal ATPase catalytic machinery in SecA [kcat(N1−263) = 0.1/min]. In the isolated N-terminal domain of SecA, IRA2 stimulates ATP turnover by NBD1 [kcat(N68) = 36.4/min]. However, ATP turnover by SecA is reduced 8-fold compared with N68 [kcat(SecA) = 4.6/min]. Presumably the IRA1 switch, present in SecA but absent from N68 (Figure 1; Karamanou