ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle - PubMed (original) (raw)
ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle
David M Smith et al. Cell. 2011.
Abstract
In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules or two ATPγS plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.
Copyright © 2011 Elsevier Inc. All rights reserved.
Figures
Figure 1. Effect of increasing ATP concentration on PAN’s ability to hydrolyze ATP and to stimulate degradation of three different types of substrates
A) The rate of ATP hydrolysis by PAN at different ATP concentrations. All data are the means of three or more independent experiments +/− SD. B) The degradation rate of the fluorogenic octapeptide (LFP) by the PAN-20S complex. The activity without added nucleotide is taken as 100% for B and C. C) The degradation rate of 14C-casein to acid-soluble peptides by the PAN-20S complex. D) The degradation rate of GFP-ssrA (monitored by loss of fluorescence) by the PAN-20S complex. Since PAN alone can unfold GFP, 20S was added in excess to ensure that unfolding was coupled to degradation.
Figure 2. PAN can bind up to 4 nucleotides per hexamer and hydrolyzes ATP even at 4°C
A) The number of bound α-32P-ATP to PAN hexamer (0.4mg/ml) was determined at different ATP concentrations at 4°C, following isolation of the nucleotide bound complex by rapid spin through a size exclusion column. The data in A and B are the means of three independent experiments +/− SD. B) The concentration of 14C-ADP that was bound to PAN with increasing concentrations of PAN using saturating 14C-ADP (1 mM). C) Bound ATP is rapidly hydrolyzed to ADP. α-32P-ATP was incubated with PAN at 4° or 25° C and the bound nucleotides were isolated into a reaction-quenching buffer, and analyzed on silica TLC plate. The image is representative of 3 independent experiments. Identical experiments using γ32P-ATP showed that the hydrolyzed Pi was released from PAN.
Figure 3. PAN contains two different types of binding sites for ATPγS and its ability to associate with the 20S and open its gate are greater with 2 ATPγS bound than with 4 bound
A) The number of ATPγS molecules bound to PAN was determined at different 35S-ATPγS concentrations at 25°C, following isolation of the complex as in Fig 2A. The data from A, B and C are the means of three independent experiments +/− SD. B) The rate of LFP hydrolysis (a measure of gate-opening) by the PAN-20S at different ATPγS concentrations. C) The rate of 14C-casein degradation to acid soluble peptides by the PAN-20S complex at different ATPγS concentrations. D) The association of PAN with the 20S proteasome, as determined by surface plasmon resonance, is greater at low ATPγS concentrations (0.01 mM) where 2 ATPγS are bound than at high concentrations (0.3mM) where 4 are bound. These curves are representative of more than 3 independent experiments.
Figure 4. ATP binding to PAN stimulates binding of protein substrates
A) Binding of FITC-casein (0.1 µM) or GFP-ssrA (0.08 µM) to PAN was monitored by fluorescence polarization in the presence of different nucleotides (1mM). B) PAN’s ability to bind a fluorescamine-labeled-ssrA peptide (0.5µM; ANDENYALAA) or an ssrA peptide with two aspartates in its C-terminus, DDssrA, (ANDENYALDD) was determined in the presence or absence of ATPγS (0.1mM). C) The change in polarization of FITC-casein (0.1 µM) by PAN at different ATPγS concentrations. Due to the high level of fluorescence intensity required for polarization assays, PAN had to be used at 1 µM to saturate binding of the FITC-casein (C and D), and thus these assays were carried out under “ligand depletion condition” (i.e. free [ATPγS] ≪ total [ATPγS]), which causes a shift in the apparent affinity of PAN for ATPγS compared to the actual affinity (Fig3). D) The change in polarization of GFP-ssrA (0.08 µM) by PAN at different ATPγS concentrations.
Figure 5. PAN functions optimally with 2 ATPγS and 2 ADP bound and gate-opening in the 26S proteasome shows similar multiphasic dependence on ATPγS as the PAN-20S complex with similar ATPγS affinities
A) 100µM of 14C ADP was mixed with different concentrations of PAN with or without 50 µM of ATPγS (2-bound state). The amount of bound 14C-ADP was determined as in Fig 2A. See also Figure S2. B) The extent of gate-opening by PAN was determined by assaying LFP hydrolysis by the PAN-20S complex in the presence of the indicated nucleotides. C) The rate of GGL-amc (20µM) hydrolysis by yeast 26S proteasomes (2µg/ml) at different ATPγS concentrations. D) The rate of suc-LLVY-amc (100µM) hydrolysis by rabbit 26S proteasome (1µg/ml) was monitored at increasing concentrations of ATPγS. See also Figure S1.
Figure 6. Nucleotide binding exchange model for the proteasomal ATPases
A) Three possible patterns by which a pair of ATP molecules can bind to a hexameric ring. B) A model describing the binding-exchange reaction for the proteasomal ATPases based on the two cooperatively-linked para-positioned subunits binding ATP. Each subunit would cycle through ATP bound, ADP bound, and nucleotide-free states. The resulting ATP hydrolysis cycle is expected to occur in the clockwise direction in the order shown. See text for rationale.
Figure 7. A sterically plausible model for how the hexameric para-positioned ATPase subunits interact with the heptameric 20S α- ring and why the 4-bound state reduces function
A) The order of the eukaryotic ATPases showing the alternating order of the HbYX and non-HbYX subunits. B) Because ATP binding to PAN drives PAN-20S association, and because only two para subunits bind ATP, it’s likely that only these two para C-termini interact with the 20S pockets at any instant. When 4 ATPγS bind, it’s likely that 4 C-termini are extended to dock with the 20S, but this form has a reduced 20S affinity, probably caused by steric problems (see 4C). C) X-ray structures demonstrate how PAN’s para positioned C-termini can dock into the 20S intersubunit pockets without steric hindrance. Because crystal structures with PAN’s C-termini are not available, we used the structure of the PAN homolog HslU as a model. The distance between carboxy groups on para C-termini (left), and the Lys66 γ- amine group in the indicated 20S intersubunit pockets (middle) are compatible as shown by manual docking HslU’s para C-termini to the 20S α-ring (right), which shows the para C-termini (green) docked into two pockets without clashes. In this mode the other (non-para) C-termini (Red) would clash with residues in the 20S. Surface rendered structures and distance calculations were generated with Pymol (DeLano Scientific).
References
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- Benaroudj N, Goldberg AL. PAN, the Proteasome Activating Nucleotidase from archaebacteria, is a molecular chaperone which unfolds protein substrate. Nature Cell Biol. 2000;2:833–839. - PubMed
- Benaroudj N, Zwickl P, Seemuller E, Baumeister W, Goldberg AL. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol Cell. 2003;11:69–78. - PubMed
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