Proteasome Allostery: Comparison
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Allostery fundamentally describes coordination and communication between distinct sites of a multipart and multifunctional protein, or riboprotein. The proteasome rivals the ribosome in size, compositional complexity, multiplicity of active sites and substrate interactions and its importance in the cellular economy. For both, the cellular consequences of its misregulation are grave. Like the ribosome, proteasomes must coordinate multiple active sites and undergo large scale conformational realignments that optimally position its components for enzymatic processing and to move substrates. The expectation that proteasomes utilize allostery is supported by two types of experimental data. The first consists of regulators that are substrates or their components. The second consists of RPs that exhibit cooperative binding to the pair of CP sites where they dock.

  • allostery
  • proteasome
  • assembly
  • degradation

1. Allostery by Substrates and Their Components

1.1. Protein Substrates

Protein substrates of the proteasome—most typically marked for degradation by conjugation of ubiquitin chain—act as homotypic regulators. During their association, translocation, unfolding and degradation, the 19S RP responds with major conformational changes, both internal and in its relationship with the CP as reviewed in [7][1]. These acrobatics have been captured by the combined application of cryoEM imaging and FRET analysis. In the basal state of the proteasome, the 19S RP ATPase ring and CP entry pore are imperfectly aligned.
Upon substrate binding, these become co-axial, facilitating substrate passage to the internal CP sites of proteolysis. The ATPase pore loops that drive translocation reconfigure from a plane to form a spiral. The 19S RP lid rotates with respect to the base; one effect of this is to reposition RPN11, which removes ubiquitin chains, closer to the substrate entering the translocation channel. The α gate of the CP is opened, promoting substrate protein entry and presumably, exit of the peptide products of degradation. Individually and in the aggregate, substrate acts to enforce conformational changes that promote its regulated degradation.

1.2. Proteolysis Active Site Inhibitors

Inhibition of proteolytic sites within the CP also allosterically influences its association with 19S RP. The best evidence showing such allosteric effect comes from an experiment in which proteasomes were reconstituted by in vivo incubation of CP and 19S RP [21][2]. In that experiment, assembly was done in the presence of ATP, with or without further addition of the inhibitor expoxomicin. The inhibitor binds tightly and specifically to the β-ring active sites of proteolysis. Removal of ATP by dialysis (which leaves expoxomicin bound) led to dissociation of the 19S RP/CP complex when expoxomicin was absent; however, the complex was preserved by bound epoxomicin.
Similar results were observed using another inhibitor, Velcade (bortezimib); in this experiment ATP was hydrolyzed enzymatically by apyrase. Both inhibitors, although structurally different, shared the capacity to protect 26S proteasome integrity against ATP depletion. As confirmation that the stabilizing activity of epoxomicin was conferred by its binding at the CP active sites of protein cleavage, the concentration dependence of the inhibitor was found to be similar for 26S stabilization and inhibition of peptidase activity.
These data were interpreted as evidence of active regulatory communication from the proteolytic sites within the CP to the CP-19S RP interface. In this view, CP active site occupancy, whether by substrates undergoing degradation or inhibitors occupying the active sites where degradation takes place, stabilizes the protein degradation machinery, thereby, supporting processive protein degradation.
Structural studies using atomic force microscopy [22][3] further support the conclusion that proteolysis at CP active sites or occupancy by inhibitors that mimic the catalytic transition state (which include epoxomicin and bortezimib) promotes gate opening. This capacity of competitors to redirect the α termini that engage 19S RP is consistent with their stabilizing effect, as described in [21][2]. High-resolution cryoEM of proteasomes treated with active site inhibitors [23][4] provided direct evidence of conformational changes propagated from CP to the top of the lid structure, a distance exceeding 150 Angstroms.

1.3. Control by Ubiquitin Chains and Ubiquitin Binding Proteins-USP14/Ubp6

Proteasomes interact actively and reciprocally with ubiquitinated substrates and do so through their ubiquitin chain receptors and deubiquitinases. Among the latter are RPN11 (aforementioned and stoichiometric) and USP14/Ubp6 (respectively mammalian and yeast orthologs), Ubp6 is substoichiometric and, when active, edits ubiquitin chains en bloc. Binding of polyubiquitinated proteins promotes α gate opening of 26S proteasomes and enhances their peptidase activity [24,25][5][6]. They do so at least in part by association via Ubp6; its association with ubiquitin chains (or ubiquitin aldehyde) activates proteasomes by promoting α gate opening [26][7], thus, stimulating peptidase activity 2- to 7-fold.
The capacity of ubiquitin chains to promote α gate opening is clearly an allosteric effect; it remains to be determined whether this interaction is mediated exclusively by Ubp6 or involves other ubiquitin chain receptors. Ubp6 has two modes that inhibit rather than stimulate degradation: enzymatic ubiquitin chain removal, which precludes or interrupts substrate capture and further processing and a non-enzymatic inhibitory effect [27][8]. Docking of Ubp6 to the proteasome, in turn, regulates Ubp6 function, strongly stimulating its catalytic deubiquitinase activity. Ubp6 binds proteasomes through a series of apparently sequential steps, initiated at Rpn1 and completed by positioning of target ubiquitin at the Ubp6 deubiquitinase active site [28,29][9][10].
Completion of docking confers on the proteasome a configuration incompetent for processive substrate degradation. After ubiquitin chain cleavage and release, proteasome conformation is presumably released from its paused inhibited state. Structural and mutagenesis data support a mechanism whereby allosteric interactions are propagated from an activation loop of Rpt1 to a series of elements of Ubp6. Through this, Ubp6, through its interactions with both ubiquitin and proteasome, provides a switch that controls the timing of proteasome activity and editing of substrate degradation marks.

1.4. Processivity of Degradation

Once engagement and degradation of substrate is initiated, this action is normally completed, cleaving substrate, shorn of ubiquitin chains, to peptides. However, that process can be interrupted, resulting in intermediate products that are only partially degraded [30,31][11][12]. Intermediate generation is favored by the presence in a substrate of two features: a folded domain that is highly resistant to unfolding and a nearby sequence tract adjacent to a folded domain that impairs pulling by the translocase and thus impairs unfolding. When the proteasome is challenged by simultaneous engagement with a tightly folded domain and a tract that frustrates the application of force, slippage from the translocase results and, in the extreme case, can cause disengagement, thereby, producing degradation intermediates [31,32][12][13].
Can this be fully understood as a purely local set of interactions within the translocation/unfoldase apparatus, thereby excluding a role for allosteric control? The data suggest otherwise: the presence of ubiquitin chains promotes processivity [33][14]. The three ubiquitin chain receptors of the proteasome, Rpn1, Rpn10 and Rpn13, have distinct effects here [34][15]. More broadly, it would be of interest to investigate the role of other proteasome components in control of processivity (e.g., USP14/Ubp6) and to perform agnostic genetic screens for proteins that control processivity.

1.5. Activation by Phosphorylation

There are numerous reports of the effects of phosphorylation on proteasomes; these are generally activating. Although the precise nature of these effects remains unclear, some are surely allosteric. Of particular interest is phosphorylation of serine 14 of the Rpn6 protein of the 19S RP. That modification is performed by cyclic AMP kinase and hormones that act through that kinase. Phosphorylation at that site accelerates the degradation of ubiquitinated protein substrates [35][16]. It will be of interest to determine the mechanism of that activation.

2. Allostery in RP/CP Assembly

Cooperative binding of primary ligands, introduced in 1965 as a concept to describe a property of tetrameric hemoglobin [36][17], has a counterpart in proteasome allostery. As described above, initial site occupancy by O2 increases the affinity of the remaining open sites of hemoglobin. This binding site interaction gives rise to the sigmoid shape of the O2 binding isotherm: loading at low saturation increase slowly, becomes progressively more efficient and flattens as saturation is approached—a sigmoid or S-shaped curve. Such a sigmoid binding isotherm is a hallmark of homotypic interaction by protein complexes that accommodate multiple substrate interactors. An initial model by Monod, Wyman and Changeux, the MWC model [37][18] sought to explain the mechanism.
This postulates two structural states in equilibrium, R of low affinity and T of high affinity. Binding of ligand perturbs the equilibrium, redirecting it from R to T. R and T in this formulation describe not merely biochemical states but conformational states as well, in both of which symmetry of the tetramer is maintained. WResearchers thus infer, according to the MWC model, two implications of homotypic allostery: sigmoid binding and conservation of two-state symmetry, toggled between low and high affinity by ligand binding. The MWC model has been elaborated, contradicted, replaced by more complex and realistic models [1][19] but remains a tractable and simple way to consider allostery. How do data and theory apply to binding of proteasome RPs by the CP?
The experiments to be described made use of Archaea proteasomes. Although these objects are taxonomically very distant, they preserve architectural and functional features of eukaryotic proteasomes, are experimentally tractable and provide results informative of the general properties of the proteasomes of all biologic Kingdoms [38][20]. The archaeal CP, like the eukaryotic CP, is of the form α7:β7:β7:α7; however, all α and all β proteins are identical. The corresponding RP, termed PAN [39][21], is also radically simpler, composed of a single protein, six copies of which form an ATPase. This functions as unfoldase and translocase. Thematically, in archaea association of PAN to CP opens its α ring.
Studies were initiated by consideration of the following question. Do 20S proteasomes with two open α gates have greater peptidase activity compared to those with one open? To address this, CP were generated that were asymmetric: one of the two α rings contained a point mutation that precludes the docking of diverse RPs [40][22]. Thus, homogeneous populations of singly and, using control symmetric CPs, doubly capped proteasomes could be made and their activities compared. The initial results were puzzling.
The RP tested was PA26, a non-ATPase hexameric 11S cap to which the gate-opening motif of the PAN ATPase (PANc) was artificially grafted. This HbYX motif, positioned at the C terminus of PANc, has the capacity to open the α gate by direct interaction [41,42][23][24]. The hybrid PA26-PANc cap conferred peptidase activity that was indistinguishable between the symmetric doubly capped and asymmetric singly capped forms. However, when a similar experiment was done using PA26 devoid of PANc, the results were different: the doubly capped form was about twice as active [40][22].
This led to a hypothesis: A CP with two open gates has twice the peptidase activity of a CP with one gate open, suggesting that peptide gate passage can be rate-limiting for activity. PA26 opens only the local gate to which it binds; however, PA26 additionally equipped with PANc opens both the proximal α gate and, remotely and allosterically, the distal gate as well. This conjecture was strengthened by consideration of the distinct modes of gate opening by PA26-PANc and PA26.
The interaction of PA26-PANc with the α ring induces a rotation in the α subunits and displaces a reverse-turn loop that stabilizes the open-gate conformation [43][25]. PA26, which lacks the PANc HbYX motif, does not cause α subunit rotation. Both are gate openers but function through different mechanisms. Importantly, only the former transmits allosteric information to the distal gate.
The hypothesis of remote distal gate opening was tested structurally by direct examination, using cryoEM [40][22]. In CP singly capped by PA26, the proximal α gate was seen to be open, but the distal gate was closed. In contrast, in CP singly capped by PA26-PANc, both gates were open. Additionally, PAN itself, with its native HbYX motif, also allosterically opened the distal gate.
Distal gate opening was seen in the presence or absence of peptide substrate; active proteolysis at the β ring sites of peptidase activity is therefore not a condition of α gate allostery. The distance between the proximal α ring site of RP binding and the distal α ring, where the regulatory effect is observed, is about 150 Ångstroms, a remarkable distance for propagation of allostery. That PA26 binding opens the proximal gate but not the distal gate of the archaea CP as independently demonstrated using an asymmetric form of an archaea CP and NMR to assess gate opening [44][26].
An independent hallmark of allostery, cooperative binding, tested the hypothesis of distal gate opening [40][22]. CP was mixed with archaea RPs (either PA26-PANc or PA26), in 3- or 4-fold stoichiometric excess of RP/CP, to reconstitute a mixture of uncapped and singly and doubly capped proteasomes. The proportion of these three forms was scored by negative stain EM. Positive cooperativity of binding, stated qualitatively, anticipates a deficit of the singly capped form, the consequence of the initial binding event enhancing the affinity of the remaining open site, compared to the open sites of an uncapped CP.
Performing an analysis across a broad range of of RP/CP ratios, as would be required to define the full binding isotherm and thereby distinguish hyperbolic from sigmoid binding, is impractical by negative stain EM. However, the null hypothesis, that binding is independent and hyperbolic, not sigmoid, makes a specific and testable prediction, a binomial distribution of the three species: P2 for doubly bound CP, 2P (1–P) for singly bound CP and (1–P)2 for unbound CP, where P is the probability of an RP binding to an individual α ring.
Therefore, the null hypothesis predicting a binomial distribution among uncapped and singly and doubly capped proteasomes can be tested and thus accepted or rejected. That statistical analysis was done using the chi square test of significance. Using PA26-PANc, a binomial distribution was rejected with high probability, demonstrating binding is not independent. It could therefore be concluded that PA26-PANc binding is cooperative. In contrast, when reconstitution was done with PA26 lacking a grafted PANc, an approximate binomial distribution among the three forms was seen, consistent with independent PA26 binding to the two sites. The results of the structural and reconstitution studies thus mutually supported a role of HbYX in propagating the allosteric signal [40][22].
Cooperative binding of a regulatory complex to CP is not confined to Archaea; 25 years ago cooperative binding of mammalian 19S RP to CP was reported [45][27]. As described above, 19S RP in subsaturating amounts was mixed with CP, and the fraction of uncapped, singly capped and doubly capped proteasomes was scored by negative stain EM. As in the more recent work with Archaea, loading of RP on CP was found to be cooperative, not independent. Conservation of cooperative binding and communication between the ends of the CP is a property conserved between at least two biologic taxonomic Kingdoms.
What is the path for passing information between α rings? CryoEM analysis has been informative of large-scale structural concomitants of allostery in proteasomes; however, in this case the path of allosteric transmission and dynamics remains elusive and lies beyond the current limitations of resolution of cryoEM. NMR has had some success in studying allosteric interaction within the CP and may be informative in addressing the question [46][28]. Rotation induced by HbYX insertion into the α ring likely initiates the chain; however, the subsequent path traverses a long and yet unmapped path.

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