ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.The chaperone ClpA is a member of the AAA ϩ protein family (ATPase-associated with various cellular activities) and catalyzes the energy-dependent degradation of proteins through interaction with the protease ClpP in Escherichia coli (1-3). Like many other AAA proteins (4), ClpA oligomerizes into a ring structure, shaping a central pore through which substrate proteins are routed into the proteolytic core ClpP. This process involves unfolding and translocation of the substrate protein and requires the consumption of ATP. AAA proteins can be grouped into class I (two ATPase domains) and class II (one ATPase domain) ATPases. The fact that some ATPases have only one AAA module whereas others seem to require two ATPase domains stimulated researchers to investigate the roles and interdependence of the two ATP-binding modules in class I AAA proteins like Hsp104 and ClpB (5-8). ClpA also features two ATPase domains, termed D1 and D2. They are highly homologous, but differences in the amino acid sequence around the conserved regions in D1 and D2 suggested that they might have a somewhat different function (9). As was shown for several other class I members, ClpA assembles into its oligomeric state only upon binding of nucleotide (10). Indeed, substituting the invariant lysine in the Walker A motif demonstrated that nucleotide binding to D1 triggers ClpA hexamerization, whereas ATP turnover is mainly catalyzed by D2 (11, 12). However, mutations in the Walker A motif also abolish or drastically decrease the affinity for ATP, making it impossible to distinguish between effects due to ATP binding and those due to ATP hydrolysis.To study the role of ATP hydrolysis in both ATPase domains uncoupled from nucleotide binding events, we designed ClpA Walker B variants that lack the ability to hydrolyze ATP in either D1 (ClpAE286A), D2 (ClpAE565A), or both domains (ClpAE286A/E565A) but still bind ATP in bot...
Vitamin B 6 is essential in all organisms, due to its requirement as a cofactor in the form of pyridoxal 5-phosphate (PLP) for key metabolic enzymes. It can be synthesized de novo by either of two pathways known as deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent. The DXP-independent pathway is the predominant pathway and is found in most microorganisms and plants. A glutamine amidotransferase consisting of the synthase Pdx1 and its glutaminase partner, Pdx2, form a complex that directly synthesizes PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate, and glutamine. The protein complex displays an ornate architecture consisting of 24 subunits, two hexameric rings of 12 Pdx1 subunits to which 12 Pdx2 subunits attach, with the glutaminase and synthase active sites remote from each other. The multiple catalytic ability of Pdx1, the remote glutaminase and synthase active sites, and the elaborate structure suggest regulation of activity on several levels. A missing piece in deciphering this intricate puzzle has been information on the Pdx1 C-terminal region that has thus far eluded structural characterization. Here we use fluorescence spectrophotometry and protein chemistry to demonstrate that the Pdx1 C terminus is indispensable for PLP synthase activity and mediates intersubunit cross-talk within the enzyme complex. We provide evidence that the C terminus can act as a flexible lid, bridging as well as shielding the active site of an adjacent protomer in Pdx1. We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx1, and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx2 and glutamine.
The ClpAP chaperone-protease complex is active as a cylindrically shaped oligomeric complex built of the proteolytic ClpP double ring as the core of the complex and two ClpA hexamers associating with the ends of the core cylinder. The ClpA chaperone belongs to the larger family of AAA+ ATPases and is responsible for preparing protein substrates for degradation by ClpP. Here, we study in real time using fluorescence and light scattering stopped-flow methods the complete assembly pathway of this bacterial chaperone-protease complex consisting of ATP-induced ClpA hexamer formation and the subsequent association of ClpA hexamers with the ClpP core cylinder. We provide evidence that ClpA assembles into hexamers via a tetrameric intermediate and that hexamerization coincides with the appearance of ATPase activity. While ATP-induced oligomerization of ClpA is a prerequisite for binding of ClpA to ClpP, the kinetics of ClpA hexamer formation are not influenced by the presence of ClpP. Models for ClpA hexamerization and ClpA-ClpP association are presented along with rate parameters obtained from numerical fitting procedures. The hexamerization kinetics show that the tetrameric intermediate transiently accumulates, forming rapidly at early time points and then decaying at a slower rate to generate the hexamer. The association of assembled ClpA hexamers with the ClpP core cylinder displays cooperativity, supporting the coexistence of interchanging ClpP conformations with different affinities for ClpA.
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