Final Assignment Review Article Fundamental Characteristics of AAA+ Protein Family Structure and Function Justin M. Miller and Eric J. Enemark Departmen

Final Assignment Review Article
Fundamental Characteristics of AAA+ Protein Family
Structure and Function

Justin M. Miller and Eric J. Enemark

Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA

Correspondence should be addressed to Eric J. Enemark;

Received 11 June 2016; Accepted 21 July 2016

Academic Editor: Baolei Jia

Copyright © 2016 J. M. Miller and E. J. Enemark. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy
derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily
(ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are
defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis.
In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using
specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with
an emphasis on pivotal aspects elucidated with archaeal proteins.

1. Molecular Machines Are
Ubiquitous in the Cell

All cells use sophisticated protein complexes that couple the
chemical energy of nucleoside triphosphate (NTP) hydrolysis
to the generation of mechanical force [1]. These complexes
operate analogously to an engine, with NTP as the fuel that is
combusted to overcome thermodynamic barriers. Based on
this similarity, these protein complexes are termed “molecu-
lar machines” [2]. A diverse set of molecular machines are
required for a myriad of cellular functions including DNA
replication [3, 4] and recombination [5], regulated proteolysis
[6–8], protein disaggregation [9–12], protein complex disas-
sembly [13], and many others.

The AAA+ superfamily of ATPases (ATPases Associated
with various cellular Activities) are critical parts of many
molecular machines [14]. AAA+ proteins catalyze the hydrol-
ysis of adenosine triphosphate (ATP) and use the derived
energy to perform mechanical work. The AAA+ domain
architecture and the underlying ATP hydrolysis mechanism
are highly conserved. Members of this family often function
as oligomers with ATPase sites at the interfaces of adjacent
subunits. Both subunits contribute residues to a bipartite
ATPase active site such that these catalytic features are

either cis- or trans-acting. These proteins participate in
many diverse cellular events, assisted by additional modules
appended to or inserted in the AAA+ domain. Archaeal
AAA+ proteins have allowed the elucidation of several critical
features of AAA+ structure, function, and mechanism. For
example, the crystal structure of Pyrobaculum aerophilum
Cdc6 provides an extremely high resolution view for how a
AAA+ protein binds nucleotide, its associated magnesium
ion, and the water molecules that complete the magne-
sium octahedral coordination sphere [15]. Here we highlight
distinguishing structural and functional features of AAA+
proteins, highlighting critical features of AAA+ structure and
function elucidated with archaeal proteins.

2. Secondary and Tertiary Structural Features
Defining the AAA+ Protein Fold

The AAA+ family is a subset of the larger P-loop protein
superfamily [16]. All P-loop family members are structurally
similar and possess a distinct / fold. The AAA+ domain
contains 200–250 amino acids with a central -sheet in
5- 1- 4- 3- 2 strand order (Figure 1(a)). The -sheet is
flanked on both sides by -helices to form a three-tiered

Hindawi Publishing Corporation
Volume 2016, Article ID 9294307, 12 pages

2 Archaea



0 1 2 3

4 5 6 7

2 1 3

4 5



Sensor 1Arg finger Sensor 2

Sensor 3

-helical bundleSecond region of



Walker-A: [T/S]

Sensor 2


Walker-A: K

Sensor 1



(b) Cdc6: ADP:Mg2+ (PDB: 1FNN)


Walker-A: K


Sensor 2



Sensor 3

Walker-A: [T/S]
Sensor 1


(c) MCM: ADP:Mg2+ (PDB: 4R7Y)

Figure 1: Features of the AAA+ ATPase domain. (a) The AAA+ – – fold topology and active site feature locations are shown in primary
sequence and secondary structure. Helices and strands within the core / fold are colored in blue and yellow, respectively. C-terminal lid
domain helices are colored in light green. (b) Active site residues are precisely positioned to bind nucleotide and Mg2+. The Mg2+ cation is
directly coordinated by the Walker-A threonine, the -phosphate of the bound nucleotide, and four water molecules. Dashed lines indicate
discussed molecular interactions (see text). The bound ADP molecule and critical active site features are shown in stick, water molecules
as light blue spheres, and the magnesium ion as a magenta sphere. (c) The ATPase site forms at subunit interfaces by residues of adjacent
subunits. The Walker-A, Walker-B, and Sensor 1 residues are positioned on the left side of the site and all reside on the same subunit (blue,
“cis-acting”), while three basic residues are located on the right side of the site from the neighboring subunit (yellow, “trans-acting”). Bound
ADP and Mg2+ are represented identical to (b). The protein topology cartoon in (a) was prepared using the TopDraw software package [42].
All structure representations in the figure were prepared with the Pymol software package [43] and PDB accession codes 1FNN [15] (b) and
4R7Y [41] (c).

– – sandwich. Features that distinguish AAA+ family
members from other P-loop NTPases include the insertion of
4 between 1 and 3 [17], the lack of an antiparallel -strand
adjacent to 5 [18], and the lack of any additional strands
directly adjacent to either 5 or 2 [16–18]. This contrasts
other P-loop family members that contain additional –
strands, such as the ABC subfamily [18, 19].

Many AAA+ proteins have a C-terminal -helical bundle
in addition to the – – core (Figure 1(a)). The functional
roles of the helical bundle are varied and include the forma-
tion of a lid over the nucleotide binding site and mediation
of subunit interactions in oligomeric protein complexes. The
position of the helical bundle relative to the – – core fold
is often nucleotide-dependent. For example, the C-terminal
bundle of the HslU protein translocase rotates 21.5∘ between
the fully open state (apo state) and closed state (ADP-
bound) with the ATP-state representing an intermediate
conformation [20].

3. Distinguishing AAA+ Primary
Sequence Motifs

The AAA+ domain contains primary sequence motifs orig-
inally used to establish the AAA family [14, 16, 17, 21]. The
AAA+ protein family was defined after the crystal structures
of N-ethylmaleimide-sensitive fusion (NSF) protein and the

subunit of the E. coli DNA polymerase III were consid-

ered alongside multiple sequence alignments and revealed
a unique region C-terminal to 4 [14, 22, 23]. We will
discuss here the signature AAA+ sequence motifs and their
functional roles.

3.1. Walker-A and Walker-B Motifs. Like all P-loop NTPases,
AAA+ proteins have Walker-A and Walker-B motif residues
that are critical for binding and hydrolyzing ATP. The Walker-
A motif consists of a GXXXXGK[T/S] sequence, where X is
any amino acid and the C-terminal residue is either threonine

Archaea 3

or serine. Structurally, this motif forms a loop between 1
and 1 within the AAA+ topology (Figures 1(a) and 1(b))
[16, 17, 21]. This feature is the canonical P-loop and is one of
the most strongly conserved sequences for AAA+ proteins.
Minor deviations include NtrC (GXXXXGK[D/E]), MCM
(GXXXXGAKS), and MoxR (GXXXXAK[T/S]) [16].

The Walker-B motif spans 3 and is characterized by the
sequence, hhhhD[D/E], where h represents any hydrophobic
residue and the C-terminal residue is aspartate or glutamate
(Figures 1(a) and 1(b)). ATP hydrolysis catalyzed by AAA+
proteins depends on the Walker-B glutamate residue at the C-
terminus of 3 (Figure 1(b)) [16]. This conserved glutamate
residue is the catalytic base that activates a water molecule
for nucleophilic attack on the -phosphate during ATP
hydrolysis (Figure 1(b)) [16, 17, 24, 25]. Mutation of the
Walker-B glutamate therefore prevents ATP hydrolysis but
still allows ATP binding [26–29]. For this reason, the Walker-
B glutamate can be mutated to glutamine or alanine to dissect
the roles of ATP binding and ATP hydrolysis in AAA+
protein activities [17, 26, 29, 30].

The X-ray crystal structure of Pyrobaculum aerophilum
Cdc6 bound to ADP and Mg2+ shows the atomic roles of
the Walker-A and Walker-B motif residues in binding ATP
[15]. The conserved Walker-A lysine residue at the start of
1 (Figure 1(a)) directly interacts with the phosphate groups
of the bound nucleotide (Figure 1(b)). The Walker-A lysine
is commonly mutated to alanine [31] to globally disrupt
nucleotide binding since this residue is so intimately involved
in nucleotide binding. A Mg2+ cation has an octahedral coor-
dination geometry that consists of the Walker-A threonine,
the -phosphate of the bound nucleotide, and four water
molecules (Figure 1(b)). Some of Mg2+-coordinating water
molecules interact directly with the Walker-B acidic residues
(Figure 1(b)). Other than water-mediated interactions, the
Walker-B residues do not directly interact with ATP or
magnesium (Figure 1(b)) [15].

3.2. Second Region of Homology. All AAA+ proteins contain
a region C-terminal to the Walker-B motif termed the Second
Region of Homology (SRH). This region spans 15–20 residues
to include part of 4, the entire 4 helix, and the loop
connecting 4 to 5 (Figure 1(a)) [17, 32]. The SRH contains
the Sensor 1 and Arginine finger motifs, both of which
are required for ATP hydrolysis. These features coordinate
nucleotide hydrolysis and propagate conformational changes
associated with nucleotide hydrolysis between subunits in
AAA+ protein complexes [17]. Due to the functional impor-
tance and the lack of SRH in other Walker-type NTPases,
this region serves as a defining characteristic of the AAA+

The Sensor 1 motif is located at the N-terminal end of the
SRH in the loop connecting 4 to 4 (Figure 1(a)). Sensor 1
is a polar residue that is most commonly asparagine but can
also be serine, threonine, or aspartate [14, 19]. It is structurally
located between the Walker-A and Walker-B motifs and
functions in concert with the Walker-B glutamate to correctly
orient the nucleophilic water molecule that undergoes attack
on the -phosphate of the bound ATP molecule [19]. As such,

Sensor 1 is critical for proper ATPase function as well as
any function that may be coupled to ATP hydrolysis. For
example, mutation of the Sensor 1 asparagine in the ATP-
dependent protease FtsH results in a loss of protease activity
even though this motif is not located in the domain that
performs proteolysis [33].

The AAA+ ATPase site is at the interface of adjacent
subunits in a protein complex (Figure 1(c)). The Walker-A/B
and Sensor 1 residues of the ATPase site are all located on
the same subunit, while the arginine finger is derived from
the neighboring subunit. For this reason, the Walker-A/B and
Sensor 1 residues are defined as “cis-acting” residues while
the arginine finger is “trans-acting.” In primary sequence,
the arginine finger is located near the C-terminal end of the
SRH and is located in the loop between 4 and 5 [17]. This
residue is nearly always an arginine, though occasionally a
lysine residue is present [19]. The name “arginine finger” is
derived from a structural similarity with GTPase activator
proteins such as the Ras-RasGAP complex, where an arginine
residue is observed in crystal structures directed into the GTP
binding site via a “finger loop” [34]. For all known structures
of AAA+ oligomers, a similar arginine residue projects into
the ATP binding and hydrolysis site of an adjacent subunit.
Based on the GTPase activator proteins, the arginine finger
forms intermolecular interactions with the -phosphate of
the bound nucleotide that may stabilize an accumulated
negative charge that occurs in the transition state during
hydrolysis [32, 35]. Mutational studies with FtsH and NtrC
conclude that the arginine finger is necessary for hydrolysis,
but not ATP binding [33, 36]. Mutation of the arginine finger
in Sulfolobus solfataricus MCM [37], Escherichia coli RuvB
[38], and others revealed similar results with an ablation of
observable ATPase activity. Mutation of the arginine finger to
glutamate in HslU not only impairs ATP hydrolysis but also
disrupts oligomerization [32, 39].

3.3. Sensor 2 and 3 Residues. Two additional active site
features have been identified from examination of AAA+
protein sequences and structures. The first is the Sensor 2
feature, which mediates conformational changes associated
with a cycle of ATP binding and hydrolysis [32]. This gener-
ally occurs through direct interaction of a Sensor 2 residue
with the -phosphate of the bound ATP molecule, which
is consistent with reports that mutations at this position
diminish nucleotide binding [17, 32, 35, 40]. Sensor 2 is
conserved in all AAA+ proteins as an arginine or lysine and
is located near the beginning of 7. AAA family members, as
opposed to AAA+ family members, generally have an alanine
residue in the Sensor 2 position [16]. Sensor 2 functions as a
cis-acting residue in AAA+ proteins that contain C-terminal
lid domains and is a trans-acting residue in proteins lacking
a canonical -helical lid domain. One example of a trans-
acting Sensor 2 is in MCM proteins, where an insertion in
the C-terminal -helical bundle disrupts the canonical lid
domain and positions the Sensor 2 arginine as a trans-acting
residue (Figure 1(c) shows Sensor 2 from an archaeal MCM)
[41]. Similarly, papillomavirus E1 lacks a canonical lid domain
and contains a trans-acting lysine residue in the Sensor 2
structural position [40].

4 Archaea

An additional sensor residue, termed Sensor 3, is present
in the structures of the AAA+ hexameric helicases papillo-
mavirus E1 [40] and MCM proteins [41, 51]. To date, this motif
has been observed as either an arginine or a histidine in E1 or
MCM, respectively (Figure 1(c)). This residue may have a role
in stabilizing the ATP-state, where the E1 structure reveals a
trans-acting arginine that reaches across the subunit interface
to interact with a Walker-B aspartate and Sensor 1 asparagine
[40]. The 3.8 Å cryo-EM structure of the eukaryotic Mcm2–
7 helicase shows that the Sensor 3 histidine is similarly
positioned to the arginine of E1, and it interacts with the
Walker-B glutamate when at the tightest interfaces [51]. This
residue may have a role in stabilizing the compact subunit
interface that is needed for the ATP-state.

3.4. N-Linker. There are defining features of the AAA+
domain N-terminal to 0 [16, 17]. This region typically
contains a conserved glycine or a similarly small residue that
forms a cap at the N-terminus followed by another family-
conserved residue [16, 17, 52]. Based on multiple sequence
alignments, Smith and coworkers have classified proteins in
groups based on the residue after the first glycine, where PRS-
and p97-like proteins have another glycine, and HslU and
the Clp ATPases have a hydrophobic residue [52]. There is
a conserved region N-terminal to this dipeptide sequence
that runs perpendicular to the -strands of the – – core
[16]. This region is referred to as the “N-linker” and both
contributes to the ATP binding pocket and serves to connect
the AAA+ domain to other domains within a protein [52].
Due to the positioning of the N-linker between domains of
a protein and directly adjacent to the ATP binding site, this
motif may play a direct role in coupling ATP hydrolysis to
conformational changes. For example, the isoleucine-glycine
N-linker of HslU interacts with the nucleotide adenine
ring [20, 49, 52]. Comparison of nucleotide-bound and
nucleotide-free HslU structures reveals a repositioning of the
isoleucine side-chain upon nucleotide binding such that the
side-chain is excluded from the ATP binding pocket and the
glycine residue changes conformer.

4. Structural Features Define the AAA+ Clades

Though all AAA+ family members contain common fea-
tures that include the Walker motifs, the Second Region
of Homology, and so forth, many of these proteins also
contain insertions of specific secondary structural elements
within or near the core – – fold. As a result, the AAA+
family members have been classified in subdivisions based
on specific sequence and structural properties [16, 19]. These
subdivisions have been thoroughly reviewed previously [16,
19], and so we will not discuss these classifications in depth.
A brief overview of each clade is provided below.

Clade 1: Clamp Loader Clade. The clamp loader clade repre-
sents the minimal AAA+ domain without any modifications
(Figures 2(a) and 2(b)) [16, 19]. As shown in Figure 2(a),
this includes the previously discussed – – sandwich with
strand order 5- 1- 4- 3- 2 and a C-terminal -helical lid
domain. Clamp loaders are conserved in bacteria, archaea,

and eukaryotes and are required to load the ring-shaped
processivity clamps that maintain continuous association
between DNA polymerases and replicating DNA [53, 54].
This family includes Replication Factor C (RFC), / DNA
polymerase III subunits, and WHIP families, where each
family possesses unique structural features outside of the core
AAA+ protein fold [44, 55, 56]. Examples include a unique
Zn cluster insertion downstream of the Walker-A motif in
bacterial / DNA polymerase III subunits [22] and a distinct
C-terminal globular domain fused to the AAA+ domain in
the WHIP family [16].

Clade 2: Initiator Clade. Members of the initiator clade
include all cellular origin recognition proteins and helicase-
loading proteins from bacteria, archaea, and eukaryotes [19].
Clade 2 is characterized by the insertion of an extra –
helix between 2 and 2 within the – – core (shown in
salmon in Figure 2(c)). The two major families within Clade
2 are the DnaA/DnaC and Orc/Cdc6 groupings, which are
derived from bacteria or archaea/eukaryotes, respectively.
Archaeal initiators and DnaA share a common domain
organization that includes an initiator-type AAA+ domain
with a C-terminal double-stranded DNA (dsDNA) binding
domain [15, 45, 57–60]. Cocrystal structures of the Orc1
DNA binding domain or the Orc1 AAA+ domain bound to
dsDNA reveal that both domains can independently bind
to and distort DNA [45, 59]. Binding interactions between
DNA and the Orc1 AAA+ domain occur through the helical
initiator-specific motif (ISM) insertion associated with Clade
2 [45, 59]. Mutations in the ISM feature significantly impair
initiator binding to origin DNA. In bacteria, DnaA assembles
around an origin of replication and initiates local melting
of duplex DNA to enable DnaC-mediated loading of the
DnaB replicative helicase. Orc and Cdc6 serve a nearly
identical role in eukaryotes and archaea to DnaA and DnaC,
respectively, to ultimately load the replicative MCM helicase
[19, 45, 61].

Clade 3: Classic Clade. Clade 3 represents a family of proteins
that are functionally related and form closed hexameric ring
structures. A defining functional feature of this family is a
shared protein remodeling function. Classic AAA+ family
members are defined by a short -helix insertion between
2 and 2 (shown in salmon in Figure 2(d)). This structural
element forms a loop that is positioned near the axial
channel of the hexameric assembly and, based on sequence
conservation and mutagenesis studies, has been proposed to
bind substrate. In contrast to the other AAA+ subfamilies,
structure-based alignments reveal that the classic clade lacks
a conserved Sensor 2 arginine residue near the beginning
of 7 [19]. Members of Clade 3 are functionally diverse,
which is a result of contributions from components outside
of the ATPase core. As such, Clade 3 can be subdivided to
include the FtsH-, katanin-, TIP49-, AFG1-, Proteasomal-,
NSF/Cdc48/Pex-, and ClpABC-families. These families are
defined by the features of domains that are N- or C-terminal
to the AAA+ core. For example, the FtsH family contains an
N-terminal protein-interaction domain and a C-terminal Zn-
protease domain. Similarly, katanin includes an N-terminal

Archaea 5



5 1 4 3 2








Clade 1



Clade 2

Orc1 (PDB: 2V1U)


Clade 3

Vps4 (PDB: 4D81)


Clade 4

E1 (PDB: 2GXA)


Clade 5



Clade 6

NtrC1 (PDB: 1NY5)


Clade 7



Figure 2: Clade-specific features of the AAA+ domain. (a) The simplest AAA+ domain is characterized by an – – topology with a C-
terminal -helical lid domain. Helices and strands within the base domain are shown as blue cylinders and yellow arrows, respectively, and
lid domain helices are represented by green cylinders. (b–h) A crystal structure of a characteristic member of each clade is shown in cartoon
representation with helices and strands colored as blue and yellow, respectively. The insertions that distinguish each clade are highlighted in
salmon. Each structure contains a bound nucleotide molecule that is shown in stick. The protein topology cartoon in (a) was prepared using
the TopDraw software package [42]. All structure representations in the figure were prepared with the Pymol software package [43] and PDB
accession codes 2CHG [44] (b), 2V1U [45] (c), 4D81 [46] (d), 2GXA [40] (e), 1KSF [47] (f), 1NY5 [48] (g), and 4R7Y [41] (h).

microtubule interaction domain and a C-terminal helix that
may support oligomerization [13].

4.1. The Pre-Sensor 1 -Hairpin Superclade. The AAA+ fam-
ilies representing Clades 4–7 constitute the “pre-Sensor 1
-hairpin” (ps1 h) superclade, where all members share a
common -hairpin insertion between 3 and 4 (Figures
2(e)–2(h)) [16, 19]. Each member of this superclade contains
the canonical AAA+ features, a ps1 h, and additional dis-
tinguishing features. Structural and biochemical studies have
shown that the ps1 h motif is positioned near the central
channel of many protein complexes [40, 41, 47, 48]. The
crystal structure of the Clade 4 papillomavirus E1 protein
bound to single-stranded DNA (ssDNA) revealed a ps1 h
that projects into the central channel and directly interacts
with DNA [40]. In contrast, the ps1 h feature is required
for interaction of RuvA with the Clade 5 RuvB protein
rather than for substrate translocation [62]. Therefore, the
functional role of the ps1 h is likely clade-dependent.

Clade 4: Superfamily III Helicase Clade. Members of Clade
4 are exclusively viral DNA helicases that are not found in
bacteria, archaea, or eukaryotes [16]. These proteins lack a
C-terminal AAA+ lid domain but contain a unique helical
bundle that is formed by elements N- and C-terminal to the

core – – domain (shown in salmon in Figure 2(e)) [19].
The Sensor 2 residue in this clade is based on structural
position rather than sequence analysis [40] and is a trans-
acting residue, in contrast to the cis-acting Sensor 2 of
clades that possess a canonical lid domain. Clade 4 family
members also contain the ps1 h insertion between 3 and
4. Superfamily III helicases form functional hexamers that
belong to the AAA+ family, in contrast to Superfamily I and
II helicases that contain tandem RecA-domains and function
as either monomers or dimers [63]. Examples of Clade 4
proteins include the SV40 large T-antigen helicase [64, 65],
papillomavirus E1 [40, 66], and the adeno-associated virus
Rep40 [67]. The X-ray crystal structure of E1 bound to ssDNA
shows that a ps1 h lysine residue of each E1 subunit forms
a salt-bridge with the DNA phosphate backbone [40, 66].
The -hairpin from each subunit differs in height to form a
staircase-like structure that correlates with the status of the
associated ATP-site [40]. This suggests a mechanism for DNA
translocation, which is expected to be common to all SF3
helicases, where ATP is sequentially hydrolyzed around the
ring to drive ps1 h movement one staircase increment at a

Clade 5: HCLR Clade. Clade 5 is the most basic member of the
ps1 h superclade because the associated -hairpin insertion

6 Archaea

is the only feature distinguishing all HCLR family members
from the clamp loader proteins of Clade 1 (shown in salmon
in Figure 2(f)) [19]. The HCLR clade name is derived from
the four families including HslU/ClpX, ClpABC-CTD, Lon,
and RuvB. The protein translocases, HslU/ClpX, ClpABC-
CTD, and Lon can be broadly grouped together based on
shared function, which causes the RuvB branch migrator to
be classified by itself. We favor the common classification of
these proteins to reflect their shared AAA+ topology. For
the protein translocases, the ps1 h may aid in polypeptide
substrate recognition rather than active translocation. In
protein translocases, the ps1 h is positioned away from the
central hexameric channel while the loop connecting 2
and 2 has been implicated in polypeptide translocation
[7, 39, 68–70]. This loop between 2 and 2 projects into
the central channel and is characterized in all polypeptide
translocases by the sequence X-Ar- -X, where X, Ar, and
are any, aromatic, or hydrophobic residues, respectively.
In contrast, the ps1 h insertion in RuvB mediates protein-
protein interactions with RuvA. Taken together, this suggests
a role other than active substrate translocation for the ps1 h
insertion in HCLR clade members.

Clade 6: Helix-2-Insert Clade. Clade 6 family members differ
from other ps1 h superclade proteins by containing an
additional -hairpin insertion in 2 that is referred to as
the helix-2-insert (h2i, shown in salmon in Figure 2(g)).
This clade includes the NtrC- and McrB-subfamilies, which
largely differ only in function. The NtrC group activates
transcription by 54-bound RNA polymerases by using ATP
hydrolysis to drive the polymerase complex from closed to
open. Clade 6 proteins that catalyze this reaction include
NtrC [36, 48] and PspF [71]. In contrast, McrB, when
associated with McrC, functions as a methylation-dependent
restriction endonuclease [72]. Although McrB does bind
ATP, it binds GTP with greater affinity, and the assembled
endonuclease complex requires GTP hydrolysis to function
[72–74]. While the role of the h2i in McrBC is not yet clear,
mutations to the NtrC h2i impair interaction with 54-bound
RNA polymerases [48]. Thus, the h2i in NtrC may mediate
protein-protein interactions similar to the ps1 h of RuvB.

Clade 7: Pre-Sensor 2 Insert Clade. The AAA+ domain
associated with Clade 7 contains ps1 h and h2i insertions
identical to Clade 6 but differs in an additional -helical
insertion after 5 (Shown in salmon in Figure 2(h)). This
insertion is located before the Sensor 2 motif and is referred to
as the pre-Sensor 2 insertion (ps-2 insertion). ps-2 insertion
places the C-terminal helical bundle in a different position
relative to the lid domain containing clades. In the ps-2
insert clade, the C-terminal helical bundle is positioned at
the backside of the – – core in contrast to the typical
configuration where the C-terminal bundle forms a lid over
the top of the – – core. This difference affects the position
of the Sensor 2 motif located in the C-terminal bundle (at
the beginning of the 7 helix, Figure 1(a)). As a result, the
Sensor 2 residue is a trans-acting active site residue in Clade 7.
Members of the ps-2 insert clade include MCM, MoxR, YifB,
and dynein.

5. Archaeal AAA+ Hexamers Share Common
Structural and Functional Features

Many AAA+ proteins share a common protein/DNA remod-
eling or degradation function where the energy of ATP
hydrolysis is coupled to translocation along polymeric sub-
strates [3, 6, 7, 14, 40, 41, 75–82]. These proteins commonly
form a closed ring that positions central channel loops to
interact with encircled DNA or polypeptide. Central channel
loop motifs include ps1 h and h2i features and other …

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