19-1
Proteasome & other proteases
Proteasome
- core complex and regulatory cap
Other proteases
- HslUV, ClpAP, ClpXP, Lon, FtsH
19-2
The proteasome
 the proteasome is a cylindrical proteasome consisting of four stacked,
seven-membered rings
 the two outer rings are alpha subunits (inactive)
 the two inner rings are beta subunits; these are proteolytically-active
 archaeal proteasome
 one type of alpha subunit
 one type of beta subunit
 eukaryotic proteasome
 seven types of alpha subunits
 seven types of beta subunits
 regulatory cap
(at least 17 subunits)
core particle (20S)
core particle (20S)
26S
regulatory particle (19S)
 the evolution of the proteasome’s subunit complexity therefore
parallels that of archaeal/eukaryal prefoldin/chaperonin
 archaeal prefoldin, 2 subunit types; archaeal chaperonin, 1-3 types
 eukaryotic prefoldin, 6 subunit types; eukaryotic CCT, 8 types
 co-evolution with substrates?
19-3
Proteasome
components
The
proteasome
regulatory
particle
19-4
Proteasome structure
active site
N
N
N
19-5
 19S cap (regulatory
complex) is only present
in eukaryotic proteasome
and its crystal structure is
unknown
 the circled ‘N’ in Fig.
(c) and (e) represent the
N-termini of archaeal and
yeast proteasome
N
 archaea has only
alpha and beta subunits
whereas eukarya has
different homologous
alpha and beta subunits
Proteasomes from eukaryotes and archaea, showing the cap complex (magenta), core complex (blue, where alpha and beta subunits are
shown), slice surface (green), active sites (white circles) and N-termini (circled ‘N’s). In (c) and (f), cyan indicates the residues
visualized that are closest to the N-termini (threonine 13 and serine 11 respectively). (a) Electron micrograph of proteasome
holoenzyme from a representative eukaryote (Xenopus laevis). (b) Medial cut-away view of the Thermoplasma acidophilum
proteasome core. The lumen is divided into three chambers, and the central chamber contains the peptidase active sites (red). (c)
Ribbon diagram of two Thermoplasma acidophilum a subunits, showing the structure of the pore. (d) Cut-away view of the
Saccharomyces cerevisiae proteasome core. (e) Ribbon diagram of two S. cerevisiae a subunits (left: Pre9/Y13; right: Pre10/Prs1). The
N-termini of these subunits are shown to occlude the channel. Adapted from Dan Finley, Encylopedia of Life Sciences.
Eukaryotic proteasome catalytic site
 active sites (shown with white circles) are on three separate
beta subunits; threonine residues are critical during catalysis
 the proteasome contains three separate proteolytic activities:
- trypsin-like (arg, lys)
- chymotrypsin-like (tyr, phe)
- post-glutamyl (glu)
 controversial: the distance between the active site thr residues is 28A, which
may determine the length of the proteolytic fragments, i.e., ~ 8 amino acids
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11S proteasome regulator
19-7
 proteasome 11S regulator also consist of
heptameric rings and bind the 20S core much
as the 19S regulatory cap does
 also called PA26, PA28 and REG
Proteasome co-crystallized
with 11S regulator particle
 binding of the 11S particle stimulates
proteasomal activity
 may facilitate product release by opening
proteasome ‘gate’
 reduction in processivity expected for an
open conformation of the exit gate may explain
the role of 11S regulators in the production of
ligands for MHC class I molecules
 11S carboxy-terminal tails provide binding
affinity by inserting into pockets on the 20S
proteasome, and 11S activation loops induce
conformational changes in alpha-subunits that
open the gate separating the proteasome
interior from the intracellular environment
Proteasome
without 11S
regulator
Proteasome
with 11S
regulator
smaller
peptides
larger
peptides
Bacterial proteasome-like proteases?
19-8
 MOST Bacteria do not possess proteins that are closely related to the proteasome, but:
 HslV is is structurally-related to proteasome
HslUV
 HslU is the regulatory particle
 HslVU is responsible for the degradation of the cell division inhibitor SulA;
its repertoire of substrates likely includes other cellular proteins
HslU
HslV
(2 rings)
em picture
inside view of
HslV protease
(active site)
structures of 2 subunits;
superimposable to the beta
subunits of the archaeal
proteasome
 Lon and FtsH have combined regulatory and protease domains into one
single polypeptide that assembles into a ring structure
 shown to have chaperone-like activity, can disassemble aggregates, and
can mediate protein degradation
Bacterial proteasomes - comparison
eukaryotic
bacterial
all
bacteria
D
archaeal
actinobacteria
only
Figure 1 Comparison of different classes of ATP-dependent
pro- teases, shown as a side-on cross-section. (A) The
eukaryotic 26S proteasome, composed of a 20S core particle
(blue a and b subunits) flanked by 19S regulatory subunits
(magenta and orange). The 19S subunits bind to the substrate
through the covalent ubiquitin modification (yellow) and
unfold it by pulling on the unstructured initiation site (bright
green). Ubiquitin is removed to be recycled during the
degradation process. (B) The bacterial protease ClpXP,
composed of rings of the protease ClpP (dark green) and the
ATPase motor ClpX (red). ClpX binds to the degradation
signal, in this case the ssrA peptide sequence (green), which
also serves as the site for the initiation of degradation. (C) The
actinobacterial proteasome, consisting of a 20S core particle
similar to that of the 26S proteasome, and a single ring of the
ATPase Mpa (purple). Mpa binds to the substrate through the
covalent Pup modification (light green). Pup has an Nterminal unstructured region, which serves as the site for the
initiation of degradation, leading to complete degradation of
Pup. (D) Archaeal proteasome is most closely related to the
eukaryotic proteasome, with core alpha and beta subunits and
Rpt-like AAA ATPase subunits (termed PAN for Proteasome
Activating Nucleotidase) involved in protein unfolding.
Kraut and Matouschek EMBO J. 2009
*
*
ClpAP, ClpXP proteases
19-9
 ClpAP and ClpXP are ATP-dependent proteases
 ClpA, ClpX are chaperones
ClpAP, ClpXP are ‘active’ proteases
ClpP by itself not active as protease
 ClpP is the protease
 substrates: soluble, abnormal proteins
ClpAP and ClpXP can also degrade any protein tagged with SsrA, an 11-residue
peptide that is added to arrested chains in bacteria
 ClpA and ClpX (in the absence of ClpP) can also disassemble protein complexes
(similar to how Hsp104 from yeast can disentangle protein aggregates)
- ClpP (2 rings) has
been crystallized
- ClpA, ClpX attach
As single rings on
Opposite sides of ClpP
 symmetry mismatch
 ClpA and ClpX have six subunits per ring; ClpP is a homo-heptameric ring
 symmetry mismatch may have implications for activity (but, other proteases
don’t have this symmetry mismatch, so relevance is not clear)
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ATP-dependent protease mechanisms
 unfolding, then degradation is a common mechanism to ATP-dependent
proteases?
 work with ClpAP, ClpXP suggest that this is the case
 PAN (Proteasome Activating Nucleotidase)
 associated with archaeal proteasome; stimulates its activity
 AAA ATPase (as with base of 19S proteasome cap); hexameric ring
 work with PAN also suggests unfolding then degradation mechanism
 the ATPase subunits of the
proteasome regulatory particle
 shown to have chaperone
activity Braun et al. (1999)
Nat. Cell Biol. 1, 221-226.
 likely also involved in
unfolding substrates just
before translocation into the
core particle
next lecture: evidence for ClpA unfolding
Compartmentalization
 compartmentalization, with respect to protein folding and degradation,
refers to the encapsulation of substrates within a cavity, or a shielded
environment
chaperones
 chaperonins possess a cavity that is capped by a cofactor (in the case of
GroEL/GroES) or with protrusions (in the case of Group II chaperonins CCT
and thermosome)
 AAA ATPases are also ring-shaped structures that possess a cavity
 prefoldin may partially envelopes substrates
 this encapsulation provides shielding of substrate hydrophobic residues
 in the case of chaperonins it provides ‘infinite dilution’ for substrates
proteases
 most oligomeric proteases have a cavity that is shielded from
the bulk cytosol
- e.g., proteasome, HslUV, ClpAP/XP, Tricorn protease, etc.
 shielding the active site is necessary to prevent
unregulated proteolysis
 encapsulation may assist processivity of protease
folding
 newly-made, non-native proteins are shielded from the bulk cytosol by chaperones
19-11