Mechanism of Homotropic Control to Coordinate Hydrolysis in a Hexameric AAA+ Ring ATPase

doi:10.1016/j.jmb.2008.05.075

J. Mol. Biol. (2008) 381, 1–12

Available online at www.sciencedirect.com

Mechanism of Homotropic Control to Coordinate Hydrolysis in a Hexameric AAA+ Ring ATPase Jörg Schumacher 1 ⁎, Nicolas Joly 1 , Inaki Leoz Claeys-Bouuaert 1 , Shaniza Abdul Aziz 1 , Mathieu Rappas 2 , Xiaodong Zhang 2 and Martin Buck 1 ⁎ 1

Division of Biology, Imperial College London, London SW7 2AZ, UK 2

Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK Received 25 March 2008; received in revised form 28 May 2008; accepted 29 May 2008 Available online 5 June 2008

AAA+ proteins are ubiquitous mechanochemical ATPases that use energy from ATP hydrolysis to remodel their versatile substrates. The AAA+ characteristic hexameric ring assemblies raise important questions about if and how six often identical subunits coordinate hydrolysis and associated motions. The PspF AAA+ domain, PspF1–275, remodels the bacterial σ54– RNA polymerase to activate transcription. Analysis of ATP substrate inhibition kinetics on ATP hydrolysis in hexameric PspF1–275 indicates negative homotropic effects between subunits. Functional determinants required for allosteric control identify: (i) an important link between the ATP bound ribose moiety and the SensorII motif that would allow nucleotide-dependent α-helical α/β subdomain dynamics; and (ii) establishes a novel regulatory role for the SensorII helix in PspF, which may apply to other AAA+ proteins. Consistent with functional data, homotropic control appears to depend on nucleotide state-dependent subdomain angles imposing dynamic symmetry constraints in the AAA+ ring. Homotropic coordination is functionally important to remodel the σ54 promoter. We propose a structural symmetry-based model for homotropic control in the AAA+ characteristic ring architecture. © 2008 Elsevier Ltd. All rights reserved.

Edited by J. Karn

Keywords: AAA+; transcription; allostery; σ54; RNA polymerase

Introduction The multi-subunit DNA-dependent RNA polymerase (RNAP) is responsible for the fundamental reaction of DNA transcription. Transcriptional initiation invariably involves RNAP-mediated opening of promoter DNA necessary to gain access to the DNA template strand used for RNA synthesis. Open complex formation of the bacterial σ54– RNAP and the eukaryotic RNAPII requires energy derived from ATP hydrolysis, provided by either the ATPases associated with various cellular activities (AAA+ domain) of the σ54 activator (also called enhancer binding proteins (EBPs)) or an ATP dependent DNA helicase, respectively.1 Remodel-

*Corresponding authors. E-mail addresses: [email protected]; [email protected]. Abbreviations used: RNAP, RNA polymerase; AAA+, ATPases associated with various cellular activities; EBP, enhancer binding protein.

ling of the σ54–promoter complex in which the promoter DNA is partially melted, termed σ54 isomerisation,2 is a necessary energy-dependent regulatory step during transcriptional activation of σ54–RNAP.3–5 The versatile AAA + proteins are molecular machines that remodel their various substrates.6,7 The AAA+ domain of PspF, PspF1–275, is necessary and sufficient to activate transcription of σ54–RNAP in vitro and in vivo.8–10 The transient energy coupling conformation was structurally captured by combining the crystal structure of hexameric PspF1–275 with the cryo-EM structure of PspF1–275 bound to the ATP transition state analogue ADP-AlFx and in complex with σ54.11 Only a subset of opposing PspF subunits show connecting densities between the hexameric PspF1–275 and σ54. Structurally conserved core elements of AAA+ proteins define their overall conserved tertiary structure, composed of one α/β subdomain and one α-helical subdomain.7 AAA+ subunits assemble front to back (heterologous association), thought to thus form hexameric ring structures (see Fig. 6a left-

0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

PspF AAAþ Hexameric Ring Coordination

2 hand panel for a hexameric PspF1–275 ring assembly). The presence of a C-terminal α-helical domain with the catalytic sensor II conserved arginine residue is a defining feature of the AAA+ protein superfamily.12 The catalytic site of AAA+ ATPases lies at the interface between two subunits with three subdomains of two adjacent subunits contributing determinants essential for hydrolysis (see Fig. 6a right-hand panel for the catalytic site and PspF1–275 interface): the sensor II conserved arginine residue (R227 in PspF) of the αhelical subdomain, the Walker B residues (D107 and E108 in PspF) of the α/β-subdomain of the same subunit and the R-finger(s) within the second region of homology of the α/β subdomain of the adjacent subunit (R162 and/or R168 in PspF). Mutations of any of these determinants largely abolishes ATPase activity in AAA+ proteins, including PspF.7,10,13 AAA+ subunits are thought to have a high degree of intersubunit cooperation.7,14 The AAA+ hexameric ring structure raises important questions of if and how ATP hydrolysis between subunits is coordinated for functional output and various models have been proposed, including concerted,15,16 sequential or semi-sequential, 17,18 and partly stochastic.19 How coordination between subunits is achieved mechanistically is not clear and may vary in different proteins, but cooperativity between subunits is likely to have a major role. Here, we investigate how cooperativity between heterogeneous nucleotide-bound subunits of PspF1–275 can be reconciled with a coordinated ATP hydrolysis cycle in the hexameric assembly. We distinguish between cooperativity as the formation of an ATP hydrolysis competent interface between two adjacent subunits and allostery, which relies on nucleotide binding-induced structural transitions that alter ATP hydrolysis at more distant sites. First, we report detailed activities of sensor II helix residue variants of PspF1–275, carrying single amino acid substitutions to alanine at positions K230 and N231 (i.e., three and four residues downstream of the AAA + highly K230A and conserved sensor II arginine), PspF1–275 N231A K230A N231A . PspF1–275 and PspF1–275 show an unPspF1–275 usual protein concentration-dependence on ATP turnover rates that is inconsistent with retaining full inter-subunit cooperativity. Next, we show PspF1–275 ATP substrate inhibition kinetics that are consistent with the theory of homotropic control between symmetrically organised but heterologously associated oligomers. Strikingly, the allosteric inhibition of PspF1–275 observed at high concentrations of ATP is K230A , indicating a role for K230 in attenuated in PspF1–275 negative homotropic control. Proximity between K230 and the bound ATP ribose hydroxyl groups (PDB 2c96)20 may contribute to relative reorientations between the α/β subdomain and the α-helical subdomain of PspF1–275. This mechanism is substantiated by the observation that dATP shows a strongly reduced negative allostery on PspF1− 275 activity compared to ATP. Because ATP outperforms dATP in isomerising the σ54–promoter complex in vitro, coordination through homotropic inhibition is functionally important for σ54-dependent transcrip-

tional activation. Our results suggest a model in which coordination between PspF1–275 subunits is achieved through nucleotide state-dependent subdomain orientations that constrain or enhance cooperativity for ATP hydrolysis in other subunits within the AAA+ ring. Negative control through allostery between adjacent ATP-bound subunits would be compatible with alternating nucleotide occupancy to maintain symmetry. Structural constraints imposed by the hexameric ring assembly may thus more generally allow a ring-specific mechanism of allosteric control in AAA+ proteins that coordinate hydrolysis and associated motions.

Results Reduced apparent cooperativity between K230A N231A and PspF1–275 subunits PspF1–275 The sensor II helix is the most highly conserved structural feature of the α-helical subdomain of AAA+ proteins.7 A direct role in catalysis was established for the highly conserved AAA+ sensor II R residue in a number of AAA+ proteins,7 including PspF (R227 in PspF).20 Structures of PspF in different nucleotide states provided evidence that two residues located within the EBP conserved sensor helix II (K230 and N231 in PspF) may be involved in nucleotidedependent inter-subunit communication. 20 The K230 side chain is proximal to the ribose groups of the bound nucleotide, while N231 lies at the subunit– subunit interface. In EBPs, the side chain at position 230 in PspF is most frequently characterised as lengthy with a charged or polar end group (the pfam family 00158 of σ54 activators shows 33% as E, 24% as R, 14% as K, 11% as Q, see Supplementary Data Fig. 4). The second residue at position 231 in PspF is predominantly charged or polar in the AAA+ family (82 out of 104 sequences aligned by Neuwald and co-workers21). To investigate the possible role of these residues in PspF, we substituted residues at positions 230 and 231 with alanine. Purified K230A proteins resulted in soluble variants, PspF1–275 N231A and PspF1–275 . Earlier isothermal calorimetric binding assays were monophasic and showed a ratio of nucleotide per polypeptide chain close to 1:1, suggesting only modest cooperativity between subunits for ATP binding to the six PspF1–275 subunits.22 To correlate ATP binding activity to ATP hydrolysis, we determined the steady-state kinetic ATP hydrolysis paraK230A N231A and PspF1–275 meters of PspF1–275, PspF1–275 (Table 1) in ATP titration experiments up to 1.5 mM ATP. Experiments were carried out at a protein concentration of 3 μM to allow activity measurements of hexameric assemblies (see below). Measurements at 23 °C allowed slowing of turnover rates for K230A N231A and PspF1–275 subsequent experiments. PspF1–275 show reduced ATP hydrolysis rates. Hill coefficients K230A N231A and PspF1–275 near 1 for PspF1–275, PspF1–275

PspF AAAþ Hexameric Ring Coordination

3

N231A Table 1. ATP hydrolysis parameters of PspF1–275, PspFK230A 1–275 and PspF1–275

ATP Protein PspF1–275 PpFK230A 1–275 PspFN231A 1–275

Vmax (min− 1) 33 (1.9) 1.6 (0.1) 11.8 (1)

dATP n

Vmax (min− 1)

1.36 ± 0.1 1.46 ± 0.1 1.45 ± 0.1

28 (0.95) 2.7 (0.4) 12.5 (1.7)

Km (mM) 0.15 (0.03) 0.3 (0.04) 0.25 (0.06)

Km (mM) 0.143 (0.016) 0.3 (0.1) 0.24 (0.09)

n 1.28 ± 0.1 1.42 ± 0.1 1.32 ± 0.1

Vmax, Km and Hill coefficients were determined at 3 μM PspF1–275, between concentrations of ATP from 0.01 mM to 1.5 mM at 23 °C. Vmax and Km values, and standard errors were calculated by non-linear regression with Graphit software (Erithacus Software Limited, version 5.0.11). Hill coefficients (n) over the nucleotide range were determined from a Hill plot (x, log [protein]; y, turnover/(1–turnover)).

suggest only modest positive cooperativity for ATP hydrolysis and provide evidence for stochastic ATP binding, in good agreement with the results of ITC experiments. To compare ATP binding between PspF1–275 and K230A N231A or PspF1–275 , we applied UV cross linking PspF1–275 32 of [α- P]ATP to PspF1–275 proteins.23,24 This nonequilibrium method allows detection of significant changes in nucleotide binding and has been applied to a number of AAA+ proteins, including PspF1–275.24–26 K230A N231A PspF1–275 and PspF1–275 show no apparent nucleotide-binding defect in UV cross-linking experiments (Supplementary Data Fig. 2B). Consistent with ATP UV cross-linking results, the reported high on/ off rates for ATP for PspF1–275 and other EBP24,27 suggest that differences in Km between PspF1–275, K230A N231A PspF1–275 and PspF1–275 are likely to reflect mainly differences in the speed of hydrolysis per se and not ATP binding. K230 and N231 are distant from the ATP phosphate groups (PDB 2C96) and therefore unlikely to affect hydrolysis directly. We considered indirect cooperative effects between subunits to account for the reduced hydrolysis rates for K230A N231A and PspF1–275 . PspF1–275 Cooperativity between PspF1–275 subunits is evident from the strong sigmoidal PspF1–275 concentration-dependence on ATP hydrolysis that correlates with the self-association range from dimers to hexamers, implying that dimeric PspF1–275 does not detectably hydrolyse ATP, and that the hexamer association allows cooperative subunit interfaces to form.22,24 We measured the apparent cooperativity K230A N231A and PspF1–275 for ATP hydrolysis between PspF1–275 subunits by plotting turnover rates expressed as a K230A N231A and PspF1–275 percentage of Vmax, against PspF1–275 concentrations between 0.01 and 3 μM (Supplementary Data Fig. 1). The less sigmoidal protein concenK230A N231A and PspF1–275 tration dependence for PspF1–275 indicate significantly reduced oligomerisationK230A and dependent cooperativity between PspF1–275 N231A PspF1–275 subunits. The Hill coefficients when plotting the relative turnover of ATP against PspF1–275, K230A N231A PspF1–275 and PspF1–275 are 2.9, 1.6, and 1.5, respectively (Supplementary Data Fig. 1B). The value of n = 2.9 for PspF1–275 is in good agreement with previously reported non-parametric bootstrap analysis of the regression coefficient in these assays, indicating high probability for n = 3.24 K230A and PspF N231A Hill coefficients for PspF1–275 1–275 suggest that their subunits cooperate approximately half as efficiently during hydrolysis or self associate less efficiently.

Distinct apo–, ADP– and ATP–PspF1–275 subunit interfaces To distinguish between defects in self-association K230A and inter-subunit cooperativity, we assayed PspF1–275 N231A and PspF1–275 for hexamer formation by size-exclusion chromatography, in the absence and in the presence of ADP or ATP. When ADP or ATP was present, we chose the lowest concentration of protein that could still be detected with satisfactory confidence to maximise the sensitivity for observing defects in hexamer formation (Fig. 1). In the absence of nucleoK230A elute as an apparent tide, PspF1–275 and PspF1–275 dimer at a 10 μM column injection concentration and as a predominantly apparent hexamer at 63.9 μM (left panel). In the presence of 0.5 mM ADP or ATP, K230A elute predominantly as PspF1–275 and PspF1–275 apparent hexamers at 10 μM. The similarities in K230A self-association between PspF1–275 and PspF1–275 suggest that the reduced cooperativity seen for K230A is not due to defects in forming hexamers. PspF1–275 N231A shows apparent defects in hexamer PspF1–275 formation in the apo- and ATP-bound forms. DifferN231A that depend on ences in selfassociation for PspF1–275 the presence and type of bound nucleotide indicate that nucleotide-free, and ADP- and ATP-bound subunits each form distinct subunit interfaces. Negative homotropic effects depend on the K230 side chain Despite necessary cooperativity between subunits of PspF1–275, the evidence for stochastic binding (see above) and ATP hydrolysis at ATP concentrations up to 1.5 mM (Table 1) suggests that subunits within the hexamer can function partly independently to bind and hydrolyse ATP. Under these conditions, random ATP hydrolysis and associated motions would not be functionally coordinated in space or time between subunits. However, we observed that ATP concentrations above 1.5 mM reduce ATP hydrolysis in PspF1– 22 We carried out ATP titration experiments up to 275. 10 mM ATP and measured ATP hydrolysis rates. We considered only time-points where between 10% and 30% of initial ATP had been hydrolysed, to obtain accurate readings and avoid effects of ATP depletion during experiments (Fig. 2a, diamonds). Hydrolysis rates were constant over time in this range. We excluded the possibility of potential unspecific chemical perturbation by ATP at high concentrations that could irreversibly inhibit PspF1–275 in time-course experiments with initial high

4

PspF AAAþ Hexameric Ring Coordination

N231A Fig. 1. Size-exclusion chromatography of PspF1–275, PspFK230A 1–275 and PspF1–275 in the absence and in the presence of N231 nucleotide. (a) Overlaid and offset chromatographs of PspF1–275 (WT), PspFK230A 1–275 and PspF1–275 at high (64 μM) and low (10 μM, grey line) concentrations of protein injected. (b) As a but in the presence of 0.5 mM ATP at a low concentration of protein (10 μM, grey line) and at a high concentration of PspFN231A 1–275 (64 μM, black line). (c) As b but in the presence of 0.5 mM ADP at a low concentration of protein (10 μM). The column was calibrated with globular proteins apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa) and carbonic anhydrase (29 kDa) as described.23

concentrations of ATP where maximal turnover rates were recovered as ATP becomes depleted (see also Fig. 3). Also, the existence of a secondary nucleotidebinding site that would act as heterotropic allosteric effector is highly unlikely, since only one ATP molecule was bound per PspF1–275 subunit at 40 mM ATP.20 Note that the ATP concentration-

Fig. 2. Apparent negative homotropic effects at concentrations of ATP above 1.5 mM and reduced negative allostery in PspFK230A 1–275 or when using dATP as triphosphonucleotide. (a) PspF1–275 (diamonds) and PspFK230A 1–275 (squares) turnover rates at increasing concentrations of ATP, expressed as a percentage of Vmax (Table 1). (b) PspF1–275 turnover rates with either ATP (diamonds) or dATP (squares).

dependent inhibition curve is sigmoidal (between 1.5 mM and 10 mM), in agreement with allostery between subunits as originally proposed for symmetrically organised oligomers.28 In contrast, the initial

Fig. 3. Distinct kinetics for PspF1–275 ATP and dATP hydrolysis rates in time-course experiments. Starting reactions by addition of ATP or dATP to give 6 mM initial concentrations, the amounts of ADP (squares) or dADP (diamonds) produced (y axis) were plotted over time (x axis). Calculating turnover rates (right-hand scale) as a function of ΔADP (open squares) or ΔdADP (open diamonds) produced between subsequent time-points (Δt), reveal different hydrolysis rate maxima for ATP or dATP that depend on triphospho nucleotide concentrations and/or diphospho to triphospho nucleotide ratios (top scale).

PspF AAAþ Hexameric Ring Coordination

rate increase (0–1.5 mM ATP) follows an apparent simple substrate-binding curve (see Discussion). These results indicate that ATP functions as a negative homotropic effector at high concentrations in the PspF1–275 hexameric ring, indicating that subunits do not hydrolyse ATP independently at higher concentrations of ATP. It seems that apparently random ATP binding to several PspF1–275 subunits allosterically inhibits ATP hydrolysis in other subunits within the hexameric assembly. To exclude the possibility that the lack of the helix-turn-helix (HTH) domain in PspF1–275 would account for our observations, we carried out ATP inhibition experiments with fulllength PspF over the same range (Supplementary Data Fig. 3). Very similar ATP inhibition rates were found, indicating that the homotropic effects of ATP are mediated through and independent of the PspF HTH domain. Because we speculated that K230 and N231 could be implicated in inter subunit commuK230A , nication, we tested previously described PspF1–275 N231A PspF1–275 and various PspF1–275 variants for ATP K230A showed reduced negainhibition.23 Only PspF1–275 tive allostery in these experiments (Fig. 2a, rectangles) compared to PspF1–275, expressed as a percentage of their individual Vmax values (Table 1). Mechanism of negative homotropic control relies on the bound ATP-ribose 2′ endo hydroxyl group The crystal structure of homogeneously ATP-bound PspF1–275 hexamer assemblies showed that the K230 side-chain NH3+ (K-NH3+ hereafter) is proximal and pointing towards the 2′-endo and 3′-exo-hydroxyl group of the bound ATP ribose (Fig. 6a left-hand panel and Discussion for structural considerations).20 We therefore tested whether 2′-endo-dehydroxyl ATP (dATP) would show reduced allostery, to determine whether the 2′-endo-hydroxyl group (2′-OH) could be a determinant in negative allostery between ATPbound PspF1–275 subunits. Plotting turnover against concentration of ATP or dATP (Fig. 2b) shows reduced negative allostery for dATP compared to ATP. These results imply that the 2′ OH group of the ATP ribose has an important role in allostery between homomeric PspF1–275 oligomers. To our knowledge, a role for 2′OH in allostery has not been reported for AAA+ proteins. Nucleoside triphosphate negative homotropic effects at high concentrations of nucleotide are predicted to be non-linear over time as ATP becomes hydrolysed. Measuring [α-32P]ATP or [α-32P]dATP turnover allows a direct determination of the relative amounts hydrolysed versus non-hydrolysed nucleotides in a time-course experiment, which has the additional technical benefit of minimising pipetting errors that may be more pronounced in titration experiments. Figure 3 shows the sigmoidal relationship between ADP or dADP produced over time with 6 mM ATP or dATP at time zero. The greater sigmoidal dependence when using ATP compared to dATP is in agreement with the higher negative allosteric effect of ATP. Plotting the turnover rates as

5 a function of ΔADP or ΔdADP produced per Δt between time-points reveals distinct optimal nucleotide compositions for hydrolysis for ATP/ADP and dATP/dADP. dATP hydrolysis is maximal at dATP concentrations between 5 mM and 3 mM where the dADP/dATP ratios are between 1:3 and 1:1. Maximal ATP turnover is reached at 1.5 mM ATP with an ADP to ATP ratio close to 3:1. These results emphasise the strong negative allosteric effect of ATP-bound PspF1–275 subunits. Higher maximal turnover rates for ATP compared to dATP may reflect a positive allosteric effect of ATP or ADP on ATP hydrolysis within the PspF1–275 hexamer at those precise nucleotide concentrations and ratios. Differences in hydrolysis rates between ATP and dATP suggest that hydrolysis is maximal when subunits are in heterogeneous nucleotide-bound states.22 However, we cannot precisely deconvolute mixed nucleotide-dependent effects, without knowledge of the exact stoichiometry of each bound nucleotide and detailed considerations of positive and negative allosteric effects as well as product inhibition. Coordination of hydrolysis through allosteric control required for productive σ54–promoter remodelling We investigated if the homotropic control impacts on transcription activation of the σ54–RNAP. Three steps during transcriptional activation by EBPs have been distinguished experimentally: (i) ATP binding induced changes resulting in contacting σ54, although these interactions are transient and difficult to measure;29 (ii) adoption of the ATP hydrolysis transition state in which EBPs engage stably with σ54, usually assessed by use of the ATP transition-state analogue ADP-AlFx;29,30 (iii) ATP hydrolysis-dependent remodelling of the σ54–promoter complex, termed σ54 isomerisation.2 Isomerisation of σ54 is necessary and probably sufficient to allow transcription activation. These features permit distinction between nucleotide binding-dependent conformational changes resulting in PspF1–275-σ54 promoter complex formation and ATP hydrolysis requiring σ54 isomerisation and transcriptional activation. We incubated a pre-formed σ54-nifH promoter probe complex with PspF1–275 variants in the presence of ADP-AlFx reagents.23 Figure 4a shows that K230A and PspFN231A can engage PspF1–275, PspF1–275 1–275 54– stably with a σ promoter complex. These results K230A N231A can bind demonstrate that PspF1–275 and PspF1–275 ADP-AlFx and adopt the transition-state conformation required to stably bind σ54–promoter DNA. In order to assess directly the capacity of PspF1–275 variants to use ATP hydrolysis to remodel the σ54– promoter complex, we carried out σ54–promoter isomerisation assays. The assay detects an open σ54– promoter complexes after isomerisation by and disengagement from PspF 1–275 . 2,31 Figure 4b K230A shows the isomerisation activities of PspF1–275 N231A N231A and PspF1–275 . Whereas PspF1–275 can isomerise K230A fails to the σ 54 –promoter complex, PspF1–275 detectably remodel its substrate. These results are

6

PspF AAAþ Hexameric Ring Coordination

the ability of ATP and dATP to isomerise the σ54– promoter complex in nucleoside triphosphate titration experiments and time-course experiments (Fig. 5). dATP and ATP have very similar turnover rates (Table 1), and is therefore more adapted to assess the importance of homotropic effects compared to K230A . ATP is clearly more able to studies with PspF1–275 support isomerisation of the σ54–promoter complex in time-course experiments and at concentrations of nucleoside triphosphate below 6 mM. Importantly, at 2 mM nucleoside triphosphate ATP has a higher isomerisation activity than dATP. Thus, functional output depends on the presence of the 2′-endohydroxyl group of the ATP ribose. Lower σ54– promoter isomerisation activity at 6 mM ATP, compared to that at lower concentrations of ATP are fully consistent with strongly reduced initial ATP hydrolysis rates at 6 mM ATP. Our results suggest that optimal σ 54 –promoter remodelling depends on homotropic control to coordinate hydrolysis between PspF1–275 subunits, which probably relies on the K– NH3+-2′-OH interactions.

Fig. 4. σ54 promoter interaction and remodelling activiN231A ties of PspF1–275, PspFK230A 1–275 and PspF1–275 . (a) Stable complex formation between PspF1–275 proteins and the σ54 promoter–DNA complex (Sinorhizobium meliloti HEX-pnifH) in the presence of non hydrolysable ADP-AlFx. Proteins, σ54 promoter–DNA complex and PspF1–275-bound σ54 promoter–DNA complexes are as indicated. (b) Activities of PspF1–275 proteins for isomerising the σ54 promoter–DNA (S. meliloti HEX-pnifH) complex by native PAGE followed by fluorescent scanning. Proteins and σ54 promoter–DNA are as indicated. Experiments are shown in the absence and in the presence of 4 mM ATP. Figures were produced by collating lanes derived from the same gels.

in complete agreement with relative activities of K230A N231A and PspF1–275 to activate transcription PspF1–275 in vitro (Supplementary Data Fig. 2). We conclude N231A are that defects in cooperativity seen for PspF1–275 probably due to the apparent defects in oligomerisation. In contrast, a lack of functional output for K230A cannot be attributed merely to defects in PspF1–275 nucleotide–dependent oligomerisation, nucleotide binding and associated motions resulting in σ54 engagement. Furthermore, low ATP turnover rates K230A are unlikely to entirely explain the of PspF1–275 lack of transcription activation activity, since turnover rates significantly lower than those seen for K230A are sufficient for functional output in PspF1–275 PspF1–275 variants that are involved directly in catalysis.23,32 To establish that the K–NH3+-2′-OH interaction is indeed required for allosteric control, we compared

Fig. 5. Performance of PspF1–275 using either ATP or dATP to isomerise the σ54–DNA promoter complex in a titration experiment (a) and in a time-course experiment (b). Reactions where carried out as described for Fig. 4b. The initial concentration of ATP was 4 mM for b. Reactions were stopped at the indicated times (b) or after 5 min (a) by loading reactions on native PAGE and applying current for 2 min to electro-elute nucleotides.

PspF AAAþ Hexameric Ring Coordination

Discussion Mechanism of subdomain motions through K–NH3+-2′-OH interactions The sigmoidal ATP inhibition kinetics on ATP turnover suggest homotropic control between ATPbound subunits is operating between subunits in hexameric PspF1–275 (Fig. 2a). Apparent negative homotropic effects depend on the presence of the K230 side chain and the ATP ribose 2′-endo group (Fig. 2a and b) and may suggest an important role for the K–NH3+-2′-OH interaction in coordination of hydrolysis between subunits in different nucleotide states. The buried position of K–NH3+-2′-OH groups excludes direct involvement of these groups in subunit–subunit contacts (PDB 2C96 and 2C98).20 The ADP-PspF 1–275 structure shows distances between the NH3+ side-chain group of K230 to the C2′-endo- and C3′-exo-hydroxyl groups of the ribose ring to be 8.3 Å and 6.6 Å, respectively. In the ATPbound PspF1–275 structure, these distances are reduced to 6 Å and 5.3 Å, mainly due to the K230 Cε–Nζ bond pivoting towards the nucleotide ribose OH groups (Fig. 6a). Although K–NH3+ and the ribose OH groups appear to be too far apart to interact directly, this is probably due to crystal constraints in the ATP-PspF1–275 structures obtained by crystal soaking. Superposition of the subunits of the EBP crystal structures of apo-PspF1–275 (PDB 2C9C),11 ADP-NtrC (PDB 1NY6)33 and ATP-ZraR (PDP 1OJL)34 show decreasing rotational α/β to αhelical subdomain angles (11° between apo-PspF1– 275 and ADP-NtrC; 18.3° between apo-PspF1–275 and ATP-ZraR). Negative stain electron microscopy and SAXS/WAXS analysis of NtrC1 further support important subdomain motions between ADP and various ATP analogues (AMPPNP, ADP-BeFx) that result in reshaping of the NtrC1 ring. Side-by-side comparisons between NtrC1 and PspF 1–275 in nucleotide type-dependent functionalities suggest that these EBPs undergo comparable nucleotide binding-induced conformational changes.29 A number of K360 side chains in the NtrC1 structure (K230 in PspF) show hydrogen bond distances with both ribose hydroxyl groups of the bound nucleotide, indicating that direct interactions are possible in the homologous NtrC1 (Supplementary Data Fig. 5). With non-constrained ATP binding-induced subdomain motions in PspF1–275, the sensor II K230 NH3+ group could readily move to within hydrogen bonding distance with the 2′-OH of ATP and thus contribute to a nucleotide binding-induced subdomain reorientation. Different nucleotide state-dependent relative subdomain orientations are evident in all AAA+ proteins for which different nucleotide state structures are available,15,17,29,35–37 suggesting that subdomain dynamics has a more general role in AAA+ protein functionality. Nucleotide bindingspecific subdomain angles in heterogeneous nucleotide-bound subunits necessarily impact on the symmetry of oligomeric proteins.

7 Symmetry-based model of coordinating hydrolysis in hexameric PspF1–275 through negative homotropic control The close relationship between allostery and symmetry in homomeric oligomeric protein assemblies was originally proposed by Monod and colleagues in 1965, and is still valid and of relevance when considering symmetrically associated homomeric proteins.38 Asymmetry in oligomeric assemblies carries an entropy penalty, in part explaining why most homomeric assemblies have evolved to associate symmetrically. In this context, we propose a model of how the nucleotide binding-induced subdomain motions described above could mechanistically account for the observed homotropic effects, by taking into account the structural constraints imposed by the particular AAA+ ring conformation. The ATP binding-induced tighter α/β to α-helical subdomain angles (Fig. 6a, red trapeze and rectangle, respectively) would limit the dynamic movements of adjacent subunits in a hexameric ring (Fig. 6b). ATP binding induced-subdomain angles in one could impair dynamics in other subunits in adopting an ATP transition state conformation required for hydrolysis.39 This would be consistent with our earlier results, where we determined the ratio of [8-14C]ADP present per PspF1–275 protomer in the hexameric PspF1–275 ADP–AlFx complex to be 0.55.24 Since ADP-AlFx mimics the transition state conformation of ATP during hydrolysis,30 not all subunits appear to be able to adopt the transition state at the same time. ATP concentrations that would result in saturating active site occupancy results in loss of ATP hydrolysis in PspF, suggesting that PspF1–275 subunits do not function independent of hydrolysis above a threshold of ATP-occupied sites. We consider that each PspF subunit may exist in the apo-, ATP- and ADP-bound states. Our model assumes nucleotide states with different but fixed angles between the αhelical and α/β subdomain with the sum of their angles equalling 360° to maintain circularity. Note that our model does not consider cooperative nucleotide binding, due to the observed moderate cooperativity for ATP binding in PspF1–275 (Hill coefficients in Table 1), but does not exclude cooperative ATP binding. Consequences of nucleotide state-dependent angles for the ring assemblies are considered by allowing for full- or no plasticity between subunit arrangements (Fig. 6b). Although full or no plasticity is very unlikely to exist in protein ring structures, consideration of those extremes illustrates the consequences of ring-specific symmetry constraints on the number of available ATP hydrolysis-competent sites. Limited plasticity in homogeneous nucleotide state AAA+ assemblies would result in open ring assemblies or, more often, in spiral assemblies, provided that subdomain rotational angles are not equal to 60° and allowing for torsion angles out of the plane of the paper (Fig. 6b, conformation a). Interestingly, many homogeneous nucleotide state AAA+ crystal structures show

8

PspF AAAþ Hexameric Ring Coordination

Fig. 6. Mechanism and model homotropic coordinated hydrolysis through nucleotide binding-induced subdomain motions in hexameric PspF1–275. (a) Hexameric PspF1–275 subunit and subdomain assembly (left), delineating the α/β subdomain (trapeze) the α-helical subdomain (rectangle) of two differently coloured adjacent subunits (green and red). Right: Close-up of the hydrolysis site between subunits with overlaid nucleotide state conformations (apo, white; ADP, blue; ATP, magenta), indicating residues discussed in the text, from three subdomains (Walker B residues D107 and E108, sensor II residues R227, K230 and N231, and trans-acting putative R-fingers R162 and R168). The proposed K230 OH– ribose interactions are shown as broken lines. (b) A model of allosteric control through nucleotide binding-induced K-NH+3 2′-OH interactions resulting in subdomain motions and its symmetry consequences on the hexameric PspF assembly (conformations a–g), assuming no plasticity or plasticity between subunits (see the text). Relative fixed angles between the schematic α/β subdomain (long black line) and the α-helical subdomain (short line) and their colour-coding are as shown in the legend. No plasticity scenario was drawn by rotating subunits according to the preceding (anticlockwise) α/β subdomain to α-helical subdomain subunit angle. In plasticity scenarios, subunits were invariably rotated 60°. Several subdomain alignments that would allow cooperative ATP hydrolysis are circled in green, non-cooperative interfaces are circled in red. Different scenarios of nucleotide occupancies and the resulting consequences for symmetry are discussed in the text.

a P65 symmetry with one subunit per asymmetric cell, indicating a spiral self-association propensity. In these cases, subunit interfaces are not necessarily

aligned precisely to allow the R-finger(s) to engage directly in catalysis. 11,40–43 The strong negative allostery at high concentrations of ATP in hexameric

PspF AAAþ Hexameric Ring Coordination

PspF1–275 could be explained by the fact that homogeneous ATP occupancy would force all subunits into a non cooperative interface conformation despite sixfold symmetry (Fig. 6b, conformation b). Either ring integrity (conformation c) or cooperative interfaces (conformation d) would be challenged at nonequimolar nucleotide occupancy, irrespective of their order within the assembly. Equimolar but nonsymmetric nucleotide state hexamer assemblies allow for 360° turns when allowing for plasticity but not for closed ring assemblies (conformation e), while plasticity, although allowing ring maintenance, would result in misaligned hydrolysis-incompetent subunit interfaces when ATP-bound subunits are not placed symmetrically (conformation f). Only alternating nucleotide states (conformation g) with twofold symmetry would allow two opposing ATP hydrolysis subunits to be effective for hydrolysis (conformation g). The AAA+ characteristic ring structure could thus impose alternate nucleotide state requirements for maximal cooperative subunit interfaces, resulting in homotropic effects when more than the optimal number of subunits are ATP-bound or their placing is not on opposite sites of the ring. This geometry is supported by the structure of hexameric ADP-AlFx bound PspF1-275 in complex with σ54, where contacting densities between the ring and σ54 lie exclusively on opposite sides of the ring.11 Limited plasticity in hexameric PspF1-275 rings would be consistent with ATP hydrolysis rates of PspF1-275 in time-course experiments (Fig. 3) that exceed dATP hydrolysis at low tri-nucleotide concentrations. Weaker subdomain motions in the dATP case, where the disrupted K– NH3+ -2′-OH interaction would reduce negative allosteric effects between subunits, is consistent with high dATP hydrolysis rates at high concentrations of dATP. Thus, coordination between subunits in protein ring assemblies does not necessarily require sophisticated molecular communication between subunits but could exploit the specific ring-imposed symmetry constraints, associated with the requirement of three subdomains to form a catalytic site for ATP hydrolysis. The low ATPase K230A may be a consequence of activity of PspF1–275 insufficient nucleotide-dependent subdomain motions required to align the three subdomains involved in cooperative ATP hydrolysis, thus linking cooperativity and allostery. Stochastic binding associated with allosteric effects causing non-independent hydrolysis was observed also for the bacterial ClpX AAA+ protease.19,44 Our mechanistic model could explain how apparent stochastic ATP binding can result in coordinated ATP hydrolysis through homotropic control. Functional performance of PspF1-275 is higher when using ATP (Fig. 5) instead of dATP, even at tri-nucleotide concentrations where hydrolysis rates for dATP are higher in ATPase assays (Fig. 2b), suggesting that coordination of hydrolysis through negative allostery between subunits in PspF1–275 is important to efficiently remodel the σ54–promoter complex during transcription initiation. A more randomly spatial hydrolysis distribution appears to

9 impair functional performance. Coordination of ATP hydrolysis could also be aided by the σ54–RNApromoter when bound to hexameric PspF1–275. However, we could not detect pronounced differences in hydrolysis kinetics of PspF1–275 (Vmax or Km) in the presence of σ 54 –promoter or σ 54 –RNAP promoter complexes in vitro (data not shown). This may suggest that the ATPase of PspF is largely uncoupled from its remodelling activity, since it is weakly, at best, stimulated by basal transcription complexes. Use of negative allostery provides one way in which otherwise independent sites for ATPase can be harnessed to work together to achieve remodelling activity. However, subtle effects of basal transcription complexes upon negative allostery or determinants of this property may exist and serve to enhance the remodelling activity of PspF. ATP ribose hydroxyl group as important functional determinants in AAA+ proteins Homotropic control in AAA+ hexameric ring assemblies could, at least in principle, operate more generally in AAA+ proteins due to their common symmetry and mode of assembly. For PspF1–275 we favour a probabilistic ATP binding that would result in constant dynamic subdomain realignments and associated motions, with only ATP-bound subunits extending the σ54 contacting GAFTGA loop.29 Due to allosteric control between adjacent subunits, hydrolysis could occur only at a particular alternate spacing of the ATP-bound subunits. Thus, PspF1–275 may coordinate hydrolysis and associated motions required for transcription activation not in a strict sequential or rotational sense but partly in a probabilistic manner through combinatorial restricted conformations competent for hydrolysis. High nucleotide on/off rates for nucleotides in PspF1–27524 with comparatively long-lived intermediate states of the σ 54 –RNAP during transcriptional initiation would allow transcriptional activation that does not require a synchronised or sequential hydrolysis cycle between subunits. Most EBP proteins have a polar or charged residue at the equivalent position to K230 in PspF1–275 (Supplementary Data Fig. 4).21 Therefore, hydrogen bond interactions between residues at this position and the ribose hydroxyl group may be commonplace. Previous studies have focussed especially on the phosphate moiety of the nucleotide and functional contributions of the nucleoside moiety have not been researched for AAA+ proteins. Using deoxyribonucleic acids for functional studies of AAA+ proteins may provide a tool for a more detailed understanding of a coordinated hydrolysis cycle and would allow testing of whether the ribose hydroxyl groupmediated subdomain motions suggested here are more generally involved in the function of AAA+ proteins. Negative homotropic control may operate also in the AAA+ proteins MCM18 and DnaB.45 MCM shows decreased ATPase activity above 7.5 mM ATP, with striking kinetic similarities to those we observe for PspF1–275 above 1.5 mM ATP (Fig. 2a). In DnaB,

10 concentrations of ATP above 3 mM strongly inhibit ATPase and helicase activity of DnaB. Of relevance, differences in functional output between ATP and dATP or mixtures thereof have, for instance, been observed in the AAA+-related heterogeneous associated oligomeric ATPases RecA and actomyosin proteins,46,47 involved in bacterial DNA replication repair and eukaryotic cardiac contraction, respectively. In RecA, the regression process, the first step of DNA repair during replication, was stimulated by dATP. Cardiac contraction by actomyosin was enhanced significantly when sub-stoichiometric amounts (10%) of dATP compared to ATP were present. Both these studies indicate an important functional role of the ribose 2′-OH group of ATP. The particular ring assembly of AAA+ proteins and tripartite hydrolysis site architecture could direct coordination of hydrolysis through homotropic control. Different AAA+ proteins may have exploited the symmetry constraint provided by the ring to coordinate ATP hydrolysis providing functionality adapted to carry out their versatile biological functions.

Materials and Methods Mutagenesis and protein purifications Plasmid pPB19 encoding Escherichia coli PspF residues 1–275 (PspF1–275 with an N-terminal His6 tag in pET28b+) was mutagenised (Quickchange Mutagenesis Kit, Stratagene) to obtain the desired single amino acid substitutions in pspF, resulting in pPB1(K230A), pPB1(N231A). PspF1–275 proteins were purified as described.24

PspF AAAþ Hexameric Ring Coordination

Gel-filtration chromatography Gel-filtration chromatography on Superdex 200 10/300 (Amersham) was as described.22 Briefly, PspF1–275 proteins were introduced at high (63 μM) or low (10 μM) protein injection concentrations to assess concentration-dependent oligomer formation of apo PspF1–275. In the presence of ATP or ADP, the column was pre-equilibrated with 20 mM Tris–HCl (pH 8.0), 50 mM NaCl, 15 mM MgCl2 ) supplemented to give final ATP or ADP concentrations of 0.5 mM. All experiments were done at least twice. σ54–promoter binding and isomerisation σ54–promoter complexes were preformed for binding and isomerisation as described.23 Briefly, the bottom strand of the nifH promoter oligomer (− 60 to +28 of the Sinorhizobium meliloti nifH promoter) was labelled with a fluorescent HEX tag, purchased from Operon AG and named WVC3-HEX. Duplex formation between the bottom strand and non-labelled top strand occurred by mixing 5μl of HEX-labelled nifH bottom strand (200 nM) and 5 μl of (400 nM) non-labelled top strand in 10 mM Tris– HCl (pH 8.0), 1 mM MgCl2. The mixture was heated to 95 °C and allowed to anneal slowly while cooling. For isomerisation assays, the top strand carries a mismatch at positions − 12/− 11 that is thought to stabilise the melted σ54–DNA structure.2,23 Final concentrations in binding to and isomerisation of the σ54–promoter complexes were 20 nM promoter DNA, 2.3 μM σ54, 3 μM PspF1–275. Binding of PspF1–275 proteins to σ54–promoter DNA was assessed by in situ formation PspF1–275-ADP-AlFx in the presence of 0.4 mM ADP, 5 mM NaF, 0.4 mM AlCl3.30 Isomerisation assays were performed in the presence of 4 mM ATP or dATP as described.32

ATP binding and hydrolysis ATP binding assays of PspF1–275 proteins by UV crosslinking was as described.24 ATPase reactions were carried out as described.23 Briefly, ATPase assays were in reaction buffer A (above) supplemented with MgCl2 to give final Mg2+ concentrations of 15 mM and incubated at 23 °C, using 0.06 μCi/μl [α-32P]ATP or [α-32P]dATP as nucleotide tracers. All nucleotides were purchased from SigmaAldrich as sodium salts, the pH was adjusted to 7 with NaOH and concentrations were determined by measuring the absorption at 259 nM and the molar extinction coefficient of 15,400 M− 1 cm− 1. Reactions were stopped by adding five volumes of 2 M formic acid. Radiolabelled [α-32P]ADP was separated from [α-32P]ATP by thin-layer chromatography and amounts were measured by phospho-imaging (Fuji Bas-1500, Tina 2.10 g software). ATP and dATP titration experiments, and all PspF1–275 titrations were designed so that only 10–30% of the initial amount of ATP was hydrolysed to ensure that the ADP produced had no measurable effect on hydrolysis rates, ATP was not depleted and non-linearity of turnover rates over time at concentrations of ATP or dATP above 2 mM was minimal. Linearity over time in hydrolysis rates up to 1.5 mM in this range was verified. Vmax, Km values and standard errors were determined at initial concentrations of 0.05–1 mM ATP or dATP, using non-linear regression provided by the Graphit software (Erithacus Software Limited, version 5.0.11). Hill coefficients, n, were estimated using linear regression of Hill plots.

Acknowledgements We thank the BBSRC for their financial support. N.J. was supported by an EMBO fellowship (ALTF 387-2005) and S.A.A. was supported by a Nuffield Vacation bursary.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2008.05.075

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