Molecular basis of tail-anchored integral membrane protein
recognition by the cochaperone Sgt2
Received for publication, December 17, 2020, and in revised form, February 4, 2021
Published, Papers in Press, February 19, 2021,
https://doi.org/10.1016/j.jbc.2021.100441
Ku-Feng Lin, Michelle Y. Fry
‡
, Shyam M. Saladi
‡
, and William M. Clemons Jr
*
From the Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA
Edited by Ursula Jakob
The targeting and insertion of tail-anchored (TA) integral
membrane proteins (IMPs) into the correct membrane is crit-
ical for cellular homeostasis. The fungal protein Sgt2, and its
human homolog SGTA, is the entry point for clients to the
guided entry of tail-anchored protein (GET) pathway, which
targets endoplasmic reticulum-bound TA IMPs. Consisting of
three structurally independent domains, the C terminus of Sgt2
binds to the hydrophobic transmembrane domain (TMD) of
clients. However, the exact binding interface within Sgt2 and
molecular details that underlie its binding mechanism and
client preference are not known. Here, we reveal the mecha-
nism of Sgt2 binding to hydrophobic clients, including TA
IMPs. Through sequence analysis, biophysical characterization,
and a series of capture assays, we establish that the Sgt2 C-
terminal domain is
fl
exible but conserved and suf
fi
cient for
client binding. A molecular model for this domain reveals a
helical hand forming a hydrophobic groove approximately 15 Å
long that is consistent with our observed higher af
fi
nity for
client TMDs with a hydrophobic face and a minimal length of
11 residues. This work places Sgt2 into a broader family of
TPR-containing cochaperone proteins, demonstrating struc-
tural and sequence-based similarities to the DP domains in the
yeast Hsp90 and Hsp70 coordinating protein, Sti1.
An inherently complicated problem of cellular homeostasis
is the biogenesis of hydrophobic integral membrane proteins
(IMPs), which are synthesized in the cytoplasm and must be
targeted and inserted into a lipid bilayer. Accounting for
25%
of transcribed genes (
1
), IMPs are primarily targeted by
cellular signal-binding factors that recognize a diverse set of
hydrophobic
α
-helical signals as they emerge from the ribo-
some (
2
–
4
). One important class of IMPs are tail-anchored
(TA) proteins whose hydrophobic signals are their single he-
lical transmembrane domain (TMD) located near the C ter-
minus and are primarily targeted posttranslationally to either
the endoplasmic reticulum (ER) or mitochondria (
5
–
9
). In the
case of the canonical pathway for ER-destined TA IMPs, each
is
fi
rst recognized by homologs of mammalian SGTA (small
glutamine tetratricopeptide repeat protein alpha) (
4
,
6
,
10
,
11
).
Common to all signal-binding factors is the need to recognize,
bind, and then hand off a hydrophobic helix. How such factors
can maintain speci
fi
city to a diverse set of hydrophobic clients
that must subsequently be released remains an important
question.
Homologs of
Saccharomyces cerevisiae
Sgt2 (small gluta-
mine-rich tetratricopeptide repeat-containing protein 2,
refered to here as ySgt2) and
Homo sapiens
SGTA (referred to
here as hSgt2 and collectively Sgt2 for simplicity) are involved
in a variety of cellular processes regarding the homeostasis of
membrane proteins including the targeting of TA IMPs (
9
,
12
–
14
), retrograde transport of membrane proteins for ubiq-
uitination and subsequent proteasomal degradation (
15
), and
regulation of mislocalized membrane proteins (MLPs) (
16
,
17
).
Among these, the role of Sgt2 in the primary pathways
responsible for targeting TA clients to the ER is best charac-
terized,
i.e.
, the fungal G
uided E
ntry of T
ail-anchored proteins
(GET) or the mammalian T
ransmembrane R
ecognition
C
omplex (TRC) pathway. In the GET pathway, Sgt2 functions
by binding a cytosolic TA client, then transferring the TA
client to the ATPase chaperone Get3 (human homolog is also
Get3) with the aid of the heteromeric Get4/Get5 complex
(human Get4/Get5/Bag6 complex) (
13
,
18
–
20
). In this pro-
cess, TA client binding to Sgt2, after hand-off from Hsp70, is
proposed as the
fi
rst committed step to ensure that ER TA
clients are delivered to the ER membrane while mitochondrial
TA clients are excluded (
3
,
13
,
21
). Subsequent transfer of the
TA client from Sgt2 to the ATP-bound Get3 induces confor-
mational changes in Get3 that trigger ATP hydrolysis,
releasing Get3 from Get4 and favoring binding of the Get3-TA
client complex to the Get1/2 receptor at the ER leading to
release of the TA client into the membrane (
22
–
26
). Deletions
of yeast GET genes (
i.e.
,
get1
Δ
,
get2
Δ
,or
get3
Δ
) cause cytosolic
aggregation of TA clients dependent on Sgt2 (
26
,
27
).
In addition to targeting TA IMPs, there is evidence that hSgt2
promotes degradation of IMPs through the proteasome by
cooperating with the Bag6 complex, a heterotrimer containing
Bag6, hGet4, and hGet5, which acts as a central hub for a diverse
physiological network related to protein targeting and quality
control (
19
,
28
–
30
). The Bag6 complex can associate with ER
membrane-embedded ubiquitin regulatory protein UbxD8,
transmembrane protein gp78, proteasomal component Rpn10c,
and an E3 ubiquitin protein ligase RNF126, thereby connecting
hSgt2 to ER-associated degradation (ERAD) and proteasomal
activity. Depletion of hSgt2 signi
fi
cantly inhibits turnover of
ERAD IMP clients and elicits the unfolded protein response
‡
These authors contributed equally to this work.
*
For correspondence: William M. Clemons Jr.,
clemons@caltech.edu
.
RESEARCH ARTICLE
J. Biol. Chem.
(2021) 296 100441
1
© 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article unde
r the CC
BY license (
http://creativecommons.org/licenses/by/4.0/
).
(
16
). Furthermore, the cellular level of MLPs in the cytoplasm
could be maintained by coexpression with hSgt2, which possibly
antagonizes ubiquitination of MLPs to prevent proteasomal
degradation (
15
,
17
). These studies demonstrate an active role of
hSgt2 in triaging IMPs in the cytoplasm and the breadth of hSgt2
clients including TA IMPs, ERAD, and MLPs all harboring one
or more TMDs. Roles for hSgt2 in disease include polyomavirus
infection (
31
), neurodegenerative disease (
27
,
32
), hormone-
regulated carcinogenesis (
33
,
34
), and myogenesis (
35
),
although the underlying molecular mechanisms are still unclear.
The architecture of Sgt2 includes three structurally indepen-
dent domains that de
fi
ne the three different interactions of Sgt2
(
Fig. 1
A
)(
12
,
36
–
39
). The N-terminal domain forms a homo-
dimer composed of a four-helix bundle with twofold symmetry
that primarily binds to the ubiquitin-like domain (UBL) of Get5/
Ubl4AforTAIMPtargeting(
36
,
40
) orinteracts with the UBLon
the N-terminal region of Bag6 (
41
) where it is thought to initiate
downstream degradation processes (
15
,
28
,
29
). The central re-
gion comprises a cochaperone domain with three repeated TPR
motifs arranged in a right-handed superhelix forming a
“
carboxylate clamp
”
for binding the C terminus of heat-shock
proteins (HSP) (
12
,
42
). The highly conserved TPR domain was
demonstrated to be critical in modulating propagation of yeast
prions by recruiting HSP70 (
27
) and may associate with the
proteasomal factor Rpn13 to regulate MLPs (
43
). More recently,
it was demonstrated that mutations to residues in the TPR
domain, which prevent Hsp70 binding, impair the loading of TA
clients onto ySgt2 (
21
), consistent with a direct role of Hsp70 in
TA IMP targeting
via
the TPR domain. The C-terminal
methionine-rich domain of Sgt2 is responsible for binding to
hydrophobic clients such as TA IMPs (
11
,
37
,
44
). Other hy-
drophobic segments have been demonstrated to interact with
Figure 1. Structural characteristics of free Sgt2 C domain.
A
,
top
, schematic of the domain organization of Sgt2.
Below
, representative sequences from a
large-scale multiple sequence alignment of the C domain: fungal Sgt2 from
S. cerevisiae
,
S. pombe
, and
C. thermophilum
and metazoan Sgt2 from
C. savignyi
,
X. laevis
, and
H. sapiens
. Protease susceptible sites on ySgt2-C identi
fi
ed by mass spectrometry are indicated by
red arrowheads
. Predicted helices of ySgt2
(
blue
) and hSgt2 (
orange
) by Jpred (
83
) and/or structure prediction are shown.
Blue
/
orange
color scheme for ySgt2/hSgt2 is used throughout the text.
Residues noted in the text are highlighted by an
asterisk
.
B
, overlay of size-exclusion chromatography traces of ySgt2-C (
blue line
), hSgt2-C (
orange line
),
ySgt2-TPR (
blue dash
), and hSgt2-TPR (
orange dash
). Traces are measured at 214 nm, baseline-corrected, and normalized to the same peak height.
C
, Far UV
CD spectrum of 10
μ
M of puri
fi
ed ySgt2-C (
blue
) and hSgt2-C (
orange
) at RT with secondary structure decomposition from BestSel (
68
).
D
,
1
H-
15
N HSQC
spectrum of ySgt2-C at 25
C. The displayed chemical shift window encompasses all N-H resonances from both backbone and side chains. The range of
backbone amide protons, excluding possible side-chain NH
2
of Asn/Gln, is indicated by pairs of
red dashed lines
.
E
,asin
D
for hSgt2-C at 25
C.
Characterization of the client-binding domain of Sgt2
2
J. Biol. Chem.
(2021) 296 100441
this domain such as the membrane protein Vpu (viral protein U)
from human immunode
fi
ciency virus type-1 (HIV-1), the TMD
of tetherin (
44
), the signal peptide of myostatin (
35
), and the N
domain of the yeast prion forming protein Sup35 (
27
). All of
these studies suggest that the C terminus of Sgt2 binds broadly to
hydrophobic stretches, yet structural and mechanistic informa-
tion for client recognition is lacking.
In this study, we provide the
fi
rst structural characterization
of the C domains from Sgt2 (Sgt2-C) and show that, in the
absence of substrate, it is relatively unstructured. We demon-
strate that a conserved region of the C domain, de
fi
ned here as
C
cons
, is suf
fi
cient for client binding. Analysis of the C
cons
sequence identi
fi
es six amphipathic helices whose hydrophobic
residues are required for client binding. Based on this, we
computationally generate an
ab initio
structural model that is
validated by point mutants and disul
fi
de cross-linking. Arti
fi
cial
clients are then used to de
fi
ne the properties within clients
critical for binding to Sgt2-C. The results show that Sgt2-C falls
into a larger STI1 family of TPR-containing cochaperones and
allow us to propose a mechanism for client binding.
Results
The
fl
exible Sgt2-C domain
Based on sequence alignment (
Fig. 1
A
), the Sgt2-C contains a
conserved core of six predicted helices
fl
anked by unstructured
loops that vary in length and sequence. Previous experimental
work suggested that this region is particularly
fl
exible, as this
domain in the
Aspergillus fumigatus
homolog is sensitive to
proteolysis (
12
). Similarly, for ySgt2-TPR-C, the sites sensitive to
limited proteolysis primarily occur within the loops
fl
anking the
conserved helices (
Fig. 1
A
,
red arrows
and
Fig. S1
B
). This
fl
exible
nature of the C-domain likely contributes to its anomalous
passage through a gel-
fi
ltration column where Sgt2-C elutes
much earlier than the similarly sized, but well-folded, Sgt2 TPR
domain (
Fig. 1
B
), as is typical for unstructured proteins [REF].
The larger hydrodynamic radius matches previous small-angle
X-ray scattering measurement of the ySgt2 TPR-C domain
that indicated a partial unfolded characteristic in a Kratky plot
analysis. The circular dichroism (CD) spectra for both homologs
suggest that the C domain and a predicted six
α
-helical
methionine-rich region of Sgt2-C (
Fig. 1
A
), hereafter referred to
as Sgt2-C
cons
, largely assume a random-coil conformation, with
40 to 45% not assignable to a de
fi
ned secondary structure
category (
Fig. 1
C
,
Fig. S1
A
)(
45
). The well-resolved, sharp, but
narrowly dispersed chemical shifts of the backbone amide pro-
tons in
1
H-
15
N HSQC spectra of Sgt2-C (
Fig. 1
,
D
and
E
) and
Sgt2-C
cons
(
Fig. S1
,
B
and
C
) indicate a signi
fi
cant degree of
backbone mobility, similar to natively unfolded proteins (
46
)
and consistent with results seen by others (
47
), further high-
lighting the lack of stable tertiary structure (
12
). Taken all
together, Sgt2-C appears to be a
fl
exible domain.
The conserved region of the C domain is suf
fi
cient for
substrate binding
We then asked if the
fl
exible Sgt2-C is the site of client
binding in the cochaperone and if so, where within this
domain is the binding region. During puri
fi
cation Sgt2-C is
susceptible to proteolytic activity being cut at several speci
fi
c
sites (
Fig. 1
A
). Proteolysis occurred primarily at Leu
327
and in
the poorly conserved N-terminal region (between Asp
235
-
Gly
258
). Given the intervening region (ySgt2 G258-L327) is
conserved (
Fig. 1
A
), it and the corresponding region in hSgt2
may mediate client binding (
Fig. 2
A
,
gray
). To test this, we
established a set of His-tagged Sgt2 constructs of various
lengths (
Fig. 2
C
,
Fig. S2
A
). These Sgt2-C truncations were
coexpressed with an MBP-tagged client, Sbh1, and binding was
detected by the presence of captured TA clients in nickel
elution fractions (
Fig. 2
B
). The TA protein Sbh1 is the yeast
homolog of the mammalian Sec61-gamma, a component of
the ER-resident Sec translocon. While the relative ef
fi
ciency of
MBP-Sbh1 capture cannot be assessed in this assay due to
differences in total protein levels (
Fig. S2
B
), we can demon-
strate the ability of a given construct to bind to the client. As
previously seen (
13
), we con
fi
rm that Sgt2-TPR-C alone is
suf
fi
cient for capturing a client (
Fig. 2
C
). As one might expect,
the C domain was also suf
fi
cient for binding the client.
Interestingly, Sgt2-C
cons
is suf
fi
cient for binding to Sbh1. Even
a minimal region of the last
fi
ve helices (referred to as
Δ
H0)
also captures Sbh1 (
Fig. 2
C
). The predicted helices in Sgt2-
C
cons
are amphipathic and their hydrophobic faces may be
used for client binding (
Fig. 2
D
).
Each of the six helices in Sgt2-C
cons
was mutated to replace
the larger hydrophobic residues with alanines, dramatically
reducing the overall hydrophobicity. For all of the helices,
alanine replacement of the hydrophobic residues signi
fi
cantly
reduces binding of Sbh1 to Sgt2-C (
Fig. 2
,
E
and
F
). While
these mutants expressed at similar levels to the wild-type (WT)
sequence, one cannot rule out that some of these changes may
affect the tertiary structure of this domain. In general, these
results imply that these amphipathic helices are necessary for
client binding since removal of the hydrophobic faces disrupts
binding. The overall effect on binding by each helix is different,
with mutations in helices 1 to 3 having the most dramatic
reduction in binding suggesting that these are more crucial for
Sgt2
–
client complex formation. It is also worth noting, as this
is a general trend, that hSgt2 is more resistant to mutations
that affect binding (
Fig. 2
F
) than ySgt2, which likely re
fl
ects
different thresholds for binding.
Molecular modeling of Sgt2-C domain
Despite the need for a molecular model, the C domain has
resisted structural studies, likely due to the demonstrated
inherent
fl
exibility. Based on the six conserved
α
-helical
amphipathic segments (
Fig. 1
A
) that contain hydrophobic
residues critical for client binding (
Fig. 2
,
D
and
E
), we expect
some folded structure to exist. Therefore, we performed
ab
initio
molecular modeling of Sgt2-C using a variety of pre-
diction methods resulting in a diversity of putative structures
(
48
–
52
). As expected, all models showed buried hydrophobic
residues as this is a major criterion for
in silico
protein folding.
Residues outside the ySgt2-C
cons
region adopted varied con-
formations consistent with their expected higher
fl
exibility.
Characterization of the client-binding domain of Sgt2
J. Biol. Chem.
(2021) 296 100441
3
Pruning these N- and C-terminal regions to focus on the
ySgt2-C
cons
region (
Fig. S3
A
) revealed a potential binding
interface for a hydrophobic substrate. Examples are seen in
Quark models (1, 4, and 6 shown), Robetta 1 and 2, and I-
TASSER 2 and 3, whereas other models had no clearly
distinguishable groove. Given the intrinsic
fl
exibility of the
Sgt2-C domain, it is possible that models without a groove are
found in the non-TMD-bound structural ensemble.
For a working model of TMD-bound ySgt2-C, we chose the
highest scoring Quark structures where a general consistent
architecture is seen (
Fig. 3
A
)(
48
). The overall model contained
a potential client-binding site, a hydrophobic groove formed by
the amphipathic helices. The groove is approximately 15 Å
long, 12 Å wide, and 10 Å deep, which is suf
fi
cient to
accommodate three helical turns of an
α
-helix,
11 amino
acids (
Fig. 3
B
).
To validate the model, we interrogated the accuracy of the
predicted structural arrangement by determining distance
constraints from cross-linking experiments. We selected four
pairs of residues in close spatial proximity and one pair far
apart based on the Quark models (
Fig. 4
A
). Calculating a C
β
–
C
β
distance between residue pairs for each model (
Fig. 4
F
), the
Quark models 1 and 3 were the most consistent with an ex-
pected distance of 9 Å or less for the close pairs. In all alter-
native models, the overall distances are much larger and
should not be expected to form disul
fi
de bonds
in vitro
if they
represent a TMD-bound state. For Robetta, a number of the
models have pairs of residues within 9 Å, and Robetta
’
s per-
residue error estimate suggests relatively high con
fi
dence in
the C
cons
region (
Fig. S3
B
).
As a control, we
fi
rst con
fi
rmed that the cysteine
–
mutant
pairs do not affect the function of ySgt2. We utilized an
in vitro
capture assay where a yeast Hsp70 homolog Ssa1
loaded with a TA client, Bos1, delivers the client to ySgt2
(
21
,
49
,
50
)(
Fig. 4
C
). Puri
fi
ed Ssa1 is mixed with detergent-
solubilized strep-tagged Bos1-TMD (a model ER TA client)
H0
EF
B
0.03 0.10 0.19 0.27 0.32
1
0.02 0.04 0.07 0.09 0.07
WT
H3A
H2A
H4A H5A
H1A
MBP-sbh1
His-
Sc
Sgt2-C
IB: MBP-sbh1
Eluate
Lysate
Pulldown: His-ySgt2-C
Rel. Efficiency
Stdev
MBP-sbh1
His-
Hs
SGTA-C
IB: MBP-sbh1
Eluate
Lysate
Rel. Efficiency
Stdev
0.63 0.21 0.27 0.55 0.67
1
0.05 0.12 0.16 0.15 0.07
WT
H2/3A
H1A
H4A H5A
H0A
A
TPR
C
95
87
222
346
313
260
327
213
219
300
ySgt2
hSgt2
Co-expression
NTA-Ni
2+
MBP-Sbh1
His-tagged
Sgt2 constructs
His-tagged
Sgt2-TA
complex
imidazole
MBP
Thr
Sbh1
Elution
y
Sgt2-C
H2
H3
H4
H5
h
Sgt2-C
H0
H1
H2/3
H4
H5
H1
D
-4.5
(Arg)
4.5
(Ile)
Kyte & Doolittle
TA: MBP-Sbh1
His- ySgt2
His-hSgt2
C
Pulldown: His-hSgt2-C
MBP-
Sbh1
TPR-C
C
cons
C
TPR-C
CC
cons
TPR-C
CC
cons
14.4
21.5
31.0
45.0
(kDa)
6.5
- 45
- 45
- 21.5
- 6.5
- 50
- 50
(kDa)
(kDa)
Figure 2. The minimal binding region of Sgt2 for client binding.
A
, diagram of the protein truncations tested for client binding that include the TPR-C
domain, C-domain (C), C
cons
,andC
cons
Δ
H0 (
Δ
H0) from ySgt2 and hSgt2. The residues corresponding to each domain are indicated, and
gray blocks
highlight
the C
cons
region.
B
, schematic of capture experiments of MBP-tagged Sbh1 separated by a thrombin (Thr) cleavage site (MBP-Sbh1) by Sgt2 variants. After
coexpression, cell pellets are lysed and NTA-Ni
2+
is used to capture His-tagged Sgt2-TPR-C.
C
, Tris-Tricine-SDS-PAGE gel (
84
) of coexpressed and puri
fi
ed MBP-
Sbh1 and His-tagged Sgt2 truncations visualized with Coomassie Blue staining.
D
, helical wheel diagrams of predicted helices (see
Fig. 1
A
)intheC
cons
domain
of ySgt2 and hSgt2. Residues are colored by the Kyte and Doolittle hydrophobicity scale (
85
).
E
, all of the hydrophobic residues (L, I, F, and M) in a predicted
helix (H0, H1, etc.) are replaced with alanines and tested for the ability to capture MBP-Sbh1. Protein levels were quanti
fi
ed by Coomassie staining. Relative
binding ef
fi
ciency of MBP-Sbh1 by ySgt2 C domain (ySgt2-C) variants was calculated relative to total amount of ySgt2-C captured (MBP-Sbh1/Sgt2-C), then
normalized to the wild-type ySgt2-C. Experiments were performed 3 to 4 times and the standard deviations are presented. Total expression levels of th
eMBP-
Sbh1 were similar across experiments as visualized by immunoblotting (IB) of the cell lysate.
F
,asin
E
but for hSgt2.
Characterization of the client-binding domain of Sgt2
4
J. Biol. Chem.
(2021) 296 100441
that contained a p-benzoyl-l-phenylalanine (BPA)-labeled
residue, Bos1
BPA,
and diluted to below the critical micelle
concentration resulting in soluble complexes of Bos1
BPA
/Ssa1.
Full-length ySgt2 variants were each tested for the ability to
capture Bos1
BPA
from Ssa1. After the transfer reaction, each
was UV-treated to generate Bos1 cross-links. Successful cap-
ture of the TA clients by ySgt2 was detected for all cysteine
variants using an anti-strep western blot and the appearance of
a Bos1
BPA
/ySgt2 cross-link band, suggesting that the muta-
tions do not affect the structure or function of ySgt2 (
Fig. 4
C
).
We and others have demonstrated that a monomeric Sgt2 is
suf
fi
cient for binding to clients (
13
). For the distance experi-
ment, each of the cysteine
–
mutant pairs was made in the more
stable monomeric variant ySgt2-TPR-C. Each variant was
coexpressed with an arti
fi
cial client
—
a cMyc-tagged BRIL
(small, four-helix bundle protein used in previous work to aid
in the crystallization of GPCRs (
51
)) with a C-terminal TMD
consisting of eight leucines and three alanines, denoted as 11
[L8], and puri
fi
ed
via
nickel-af
fi
nity chromatography in
reducing buffer (
Fig. S4
A
). All of the ySgt2 mutants bound to
the client and behaved similar to the WT (cysteine-free)
further suggesting that the mutants did not perturb the native
structure (
Fig. S4
B
). For disul
fi
de cross-link formation, each
eluate was oxidized, digested using the protease Glu-C, and
cross-links were identi
fi
ed by the visualization of a reducing-
agent sensitive
7.7 kDa fragment in gel electrophoresis
(
Fig. 4
D
). For both the WT construct and in N285C/G329C,
where the pairs are predicted from the Quark models to be too
distant for disul
fi
de bond formation, no higher-molecular-
weight band was observed. For the remaining pairs that are
predicted to be close enough for bond formation, the 7.7 kDa
fragment was observed in each case and is labile in reducing
conditions. Again, these results support the C
cons
model
derived from Quark.
With the four cross-linked pairs as distance constraints, new
models were generated using Robetta with a restraint on the
corresponding pairs of C
β
atoms less than 9 Å (
Fig. S5
A
). The
Robetta models from these runs are similar to the top scoring
models from Quark (
Fig. 3
). Satisfyingly, the pair of residues
that do not form disul
fi
de cross-links are generally consistent
(
Fig. S5
B
).
The improvement of the ySgt2 models predicted by Robetta
with restraints included encouraged us to generate models for
hSgt2-C with constraints. For this, pairs were de
fi
ned based on
sequence alignments of Sgt2 (
Fig. 1
A
) and used as restraints.
The resulting predictions had architectures consistent with the
equivalent regions predicted for ySgt2-C
cons
, for example,
Robetta 4 (
Fig. S5
C
, top). Although in general the predicted
hSgt2 model is similar to that for ySgt2, the region that cor-
responds to H2 occupies a position that precludes a clear
hydrophobic groove. For ySgt2, the longer N-terminal loop
occupies the groove preventing the exposure of hydrophobics
to solvent (
Fig. 3
C
,
gray
). For hSgt2, the shorter N-terminal
loop may not be suf
fi
cient to similarly occupy the groove and
allow for the clear hydrophobic hand seen for the ySgt2-C. To
correct for this, we replaced the sequence of the N-terminal
loop of hSgt2-C with the ySgt2-C loop and ran structure
prediction with the pairwise distance restraints. This resulted
in a model where the loop occupies the groove and, when
pruned away, suggests the hydrophobic hand seen in yeast
(
Fig. S5
C
, middle boxed). Of note, we also generated models of
hSgt2-C using the most recent Robetta method (transform-
restrained), which produces new structures with a groove and
similar helical-hand architecture across the board (
Fig. S5
C
,
bottom).
We sought to further test the robustness of our model
considering the intrinsic
fl
exibility of Sgt2-C by probing for
disul
fi
de bond formation with neighboring residues of one of
our cross-linking pairs. While the C
β
–
C
β
distance puts these
adjacent pairs at farther than 9 Å, mutating residues to cystines
and measuring S
–
S distances across all possible pairs of
rotamers provide a wider interval on possible distances and,
therefore, the likelihood that a disul
fi
de bond will form
(
Fig. 4
E
). Cysteine mutants were introduced to the residues
adjacent to M289 and A319 in ySgt2-TPR-C resulting in four
additional pairs: K288C/A319C, M290C/A319C, M289C/
P318C, and M289C/L320C. As described previously, these
mutants were coexpressed with a substrate, in this case the
cMyc-tag was replaced with an MBP-tag. The MBP-tag on the
arti
fi
cial client allows for tandem amylose- and nickel-af
fi
nity
chromatography to ensure eluates contained only Sgt2-TPR-
C bound to substrate. Disul
fi
de bond formation was conducted
Figure 3. A structural model for Sgt2-C
cons
.
A
, the top ten models of the ySgt2-C
cons
generated by the template-free algorithm Quark (
48
) are overlaid
with the highest scoring model in solid. Models are color-ramped from N- (
blue
) to C terminus (
red
).
B
, a model of ySgt2-C
cons
(surface colored by Kyte
–
Doolittle hydrophobicity) bound to a TMD (
purple helix
) generated by rigid-body docking through Zdock (
80
). The
darker purple
corresponds to an 11-
residue stretch.
C
, the entire ySgt2-C from the highest scoring model from Quark (C
cons
in rainbow with the rest in
gray
) highlighting H0 and the rest
of the
fl
exible termini that vary considerably across models.
Characterization of the client-binding domain of Sgt2
J. Biol. Chem.
(2021) 296 100441
5
L327
M323
A319
N322
G329
I286
A272
M289
N285
Crosslinked
peptides
No bond formed
Intramolecular disulfide bond
1.1
3.5
6.5
14.2
(kDa)
-ME
A272C
L327C
I286C
M323C
M289C
A319C
M289C
N322C
−
+
−
+
−
+
−
+
−
+
−
+
N285C
G329C
WT
A
F
N285-
G329
A272-
L327
I286-
M323
M289-
A319
M289-
N322
C
-C
Distance (Å)
9 Å or less
N285-
G329
A272-
L327
I286-
M323
M289-
A319
M289-
N322
1
10.1
9.17.07.46.2
44.0
28.3
30.7
24.2
31.0
1
2
6.5
9.1
11.6
15.0
12.4
23.7
11.1
17.1
18.9
21.1
2
3
15.7
6.0
6.2
6.8
9.2
45.1
39.6
31.6
24.6
33.3
3
4
6.7
10.1
14.1
19.4
16.0
16.8
15.7
8.2
12.8
12.9
4
5
15.6
13.5
4.9
8.3
10.1
22.9
9.8
8.6
11.5
13.2
5
611.3
8.5
14.6
22.9
19.4
12.8
10.4
10.6
6
716.2
7.2
5.8
8.7
9.8
23.0
22.0
20.5
29.3
29.3
7
8
7.6
11.6
10.3
14.4
10.7
13.8
10.7
10.7
8
9
8.8
18.6
10.7
12.5
10.8
43.3
26.1
25.6
20.3
26.5
10
6.9
14.2
11.1
14.2
10.7
17.2
12.2
7.4
16.9
13.0
117.6
7.8
8.4
13.9
15.7
8.6
7.1
9.8
13.3
11.0
1
2
21.3
9.6
11.5
17.9
17.9
14.1
13.3
9.1
20.8
14.1
2
3
24.6
12.4
14.8
17.8
21.3
18.1
5.6
6.3
13.2
13.1
3
4
20.2
11.1
12.
119.921.0
8.8
9.8
9.9
10.0
7.6
4
5
17.6
10.0
6.8
17.7
17.5
11.0
7.6
8.6
12.7
10.9
5
Robetta
Pcons
I-TASSER
Quark
Phyre2
RaptorX
D
A272C
L327C
I286C
M323C
M289C
A319C
M289C
N322C
−
+
−
+
−
+
−
+ −
+
−
+
N285C
G329C
WT
20
25
37
50
75
Sgt2-Bos1
complex
Bos1 dimer
Free Bos1
Sgt2-FL
100
(kDa)
Sgt2-FL
Bos1
Ssa1
1 min
Freeze & UV treated
1 min
diluted
to 0.1uM
diluted
to 4uM
diluted
to 0.3uM
+ ATP
B
C
1.1
3.5
6.5
14.2
(kDa)
-ME
K288C
A319C
M290C
A319C
M289C
P318C
−
+
−
+
−
+
−
+ −
+
−
+
N285C
G329C
WT
M289C
A319C
M289C
L320C
−
+
E
Crosslinked
peptides
9.8-14.1 4.3-9.9 9.3-13.1 9.4-12.9 9.8-12.6 7.9-11.1
0/9 7/9 0/9 0/9 0/9 4/9
min - max range (
Å)
# pairs <9
Å
Figure 4. Validating the structural model with disul
fi
de bond formation.
Variants of His-ySgt2-TPR-C (WT or cysteine double mutants) were coexpressed
with the arti
fi
cial client, cMyc-BRIL-11[L8]. After lysis, ySgt2-TPR-C proteins were puri
fi
ed, oxidized, then digested by Glu-C protease, and analyzed by gel in
either nonreducing or reducing buffer.
A
,C
α
ribbon of ySgt2-C
cons
color-ramped with various pairs of cysteines highlighted.
Scissors
indicate protease
cleavage sites resulting in fragments less than 3 kDa in size.
B
, a schematic of the transfer of Bos1
BPA
from Ssa1 to full-length ySgt2 to demonstrate that the
double cysteine mutants are still functional.
C
, a western blot visualizing cross-linked ySgt2-Bos1 complexes. All samples tested, WT, N285C/G329C, A272C/
L327C, I286C/M323C, M289C/A319C, and M289C/N322C, had a higher molecular weight appearing after the addition of ySgt2, which corresponds to the
size of the cross-linked complex (
86
).
D
, for the WT (cys-free), no signi
fi
cant difference was found between samples in nonreducing
versus
reducing
conditions. All close residue pairs (A272/L327, I286/M323, M289/A319, and M289/N322) show peptide fragments (higher MW) sensitive to the reducing
agent and indicate disul
fi
de bond formation (indicated by
arrow
). A cysteine pair (N285/G329) predicted to be far apart by the model does not result in the
higher MW species.
E
, tris-Glycine SDS-PAGE gel probing the
fl
exibility of ySgt2-C
cons
. All new pairs (K288/A319, M290/A319, M289/P318, M289/L320) show
peptide fragments sensitive to the reducing agent (indicated by
arrow
). The range of distances of the eight closest possible rotamer pairs is annotated
below. The cysteine pair (N285/G329) shown to be far apart by the model does have a faint higher-molecular-weight band.
F
,C
β
–
C
β
distances between the
residues mutated to cysteines based on various models predicted by the Quark, I-TASSER, PCONS, and Robetta. Cysteine pairs that are 9 Å or less colored
in
orange and are expected to be close enough to form disul
fi
de bonds. Where all
fi
ve pair distances are consistent with the experiment (four near and one
far), the row is shaded in
gray
.
Characterization of the client-binding domain of Sgt2
6
J. Biol. Chem.
(2021) 296 100441
as before, and a reductant sensitive band at 7.7 kDa is observed
for each of these adjacent pairs. While the geometry of each of
these C
–
C pairs might suggest against disul
fi
de bond forma-
tion, given the intrinsic
fl
exibility of Sgt2-C, it is not surprising
that each of these pairs is able to form disul
fi
de bonds. As
before, disul
fi
de bond formation was detected for the M289C/
A319C pair. In this new construct, we now see a small amount
of disul
fi
de bond formation in the distant N285C/G329C pair,
likely an effect of switching to the MBP tag.
Structural similarity of Sgt2-C domain to STI1 domains
Attempts to glean functional insight for Sgt2-C from Basic
Local Alignment Search Tool searches did not reliably return
other families or non-Sgt2 homologs making functional
comparisons dif
fi
cult. A more extensive pro
fi
le-based search
using hidden Markov models from the SMART database (
52
)
identi
fi
ed a similarity to domains in the yeast cochaperone Sti1
(HOP in mammals). First called DP1 and DP2, due to their
prevalence of aspartates (D) and prolines (P), these domains
have been shown to be required for client binding by Sti1 (
53
,
54
) and are termed
“
STI1
”
domains in bioinformatics data-
bases (
52
). In yeast Sti1 and its human homolog HOP (com-
bination will be referred to here as Sti1), each of the two STI1
domains (DP1 and DP2) is preceded by Hsp70/90-binding
TPR domains, similar to the domain architecture of Sgt2.
Deletion of the second, C-terminal STI1-domain (DP2) from
Sti1
in vivo
is detrimental, impairing native activity of the
glucocorticoid receptor (
53
).
In vitro
, removal of the DP2
domain from Sti1 results in the loss of recruitment of the
progesterone receptor to Hsp90 without interfering in Sti1-
Hsp90 binding (
55
). These results implicate DP2 in binding
of Sti1 clients. In addition, others have noted that, broadly,
STI1 domains may present a hydrophobic groove for binding
the hydrophobic segments of a client (
53
,
54
). Furthermore,
the similar domain organizations (
i.e.
, Sgt2 TPR-C, Sti1 TPR-
STI1) and molecular roles could imply an evolutionary rela-
tionship between these cochaperones. Indeed, a multiple
sequence alignment of the Sgt2-C
cons
with several yeast STI1
domains (
Fig. 5
A
) reveals strong conservation of structural
features. H1
–
H5 of the predicted helical regions in C
cons
align
directly with the structurally determined helices in the DP2
domain of Sti1; this includes complete conservation of helix-
breaking prolines and close alignment of hydrophobic resi-
dues in the amphipathic helices (
53
).
Based on the domain architecture and homology, a direct
comparison between the STI1 domain and Sgt2-C
cons
can be
made. A structure of DP2 solved by solution NMR reveals that
the
fi
ve amphipathic helices assemble to form a
fl
exible helical
hand with a hydrophobic groove (
53
). The lengths of the
α
-
helices in this structure concur with those inferred from the
alignment in
Figure 4
A
. Our molecular model of Sgt2-C
cons
is
strikingly similar to this DP2 structure (
Fig. 5
,
B
and
C
). An
overlay of the DP2 structure and our molecular model dem-
onstrates both Sgt2-C
cons
and DP2 have similar lengths and
Figure 5. Comparison of Sti1 domains and the Sgt2-C
cons
model.
A
, multiple sequence alignment of Sgt2-C with STI1 domains (DP1, DP2) from STI1/Hop
homologs. Helices are shown based on the Sgt2-C
cons
model and the
Sc
Sti1-DP1/2 structures. Species for representative sequences are from
S. cerevisiae
(
Scer
),
S. pombe
(
Spom
),
C. thermophilum
(
Cthe
),
C. savignyi
(
Csav
), and
H. sapiens
(
Hsap
).
B
,C
α
ribbon of
Sc
Sgt2-C
cons
color-ramped with large hydrophobic
sidechains shown as
gray sticks
(sulfurs in
yellow
).
C
, similar to
B
for the solution NMR structure of Sti1-DP2
526
–
582
(PDBID: 2LLW) (
53
).
D
, superposition of the
Sgt2-C
cons
(
blue
) and Sti1-DP2
526
–
582
(
red
) drawn as cartoons.
Characterization of the client-binding domain of Sgt2
J. Biol. Chem.
(2021) 296 100441
7
arrangements of their amphipathic helices (
Fig. 5
D
). Consis-
tent with our observations of
fl
exibility in Sgt2-C
cons
, Sti1-DP2
generates few long-range Nuclear Overhauser effects between
its helices indicating that Sti1-DP2 also has a
fl
exible archi-
tecture (
53
). We consider this
fl
exibility a feature of these
helical hands for reversible and speci
fi
c binding of a variety of
clients.
Binding mode of clients to Sgt2
We examined the Sgt2-C
cons
surface that putatively in-
teracts with clients by constructing hydrophobic-to-charge
residue mutations that are expected to disrupt capture of cli-
ents by Sgt2. Similar to the helix mutations in
Figure 2
,
E
and
F
, the capture assay was employed to establish the relative
effects of individual mutations. A baseline was established
based on the amount of the TA client Sbh1 captured by WT
Sgt2-TPR-C. In each experiment, Sbh1 was expressed at the
same level; therefore, differences in binding should directly
re
fl
ect the af
fi
nity of Sgt2 mutants for clients. In all cases,
groove mutations from hydrophobic to aspartate led to a
reduction in client binding (
Fig. 6
). The effects are most dra-
matic with ySgt2 where each mutant signi
fi
cantly reduced
binding by 60% or more (
Fig. 6
A
). While all hSgt2 individual
mutants saw a signi
fi
cant loss in binding, the results were more
subtle with the strongest, a
36% reduction (M233D,
Fig. 6
B
).
Double mutants were stronger with a signi
fi
cant decrease in
binding relative to the individual mutants, more re
fl
ective of
the individual mutants in ySgt2. As seen before (
Fig. 2
,
E
and
F
), we observe that mutations toward the N terminus of Sgt2-C
have a stronger effect on binding than those later in the
sequence, whether single-point mutants in the case of ySgt2 or
double mutants for hSgt2.
Sgt2-C domain binds clients with a hydrophobic segment
≥
11
residues
With a molecular model for ySgt2-C
cons
and multiple lines
of evidence for a hydrophobic groove, we sought to better
understand the speci
fi
c requirements for TMD binding. TMD
clients were designed where the overall (sum) and average
(mean) hydrophobicity, length, and the distribution of hydro-
phobic character were varied in the TMDs. These arti
fi
cial
TMDs, a Leu/Ala helical stretch followed by a Trp, were
constructed as C-terminal fusions to the soluble protein BRIL
(
Fig. 7
A
). The total and mean hydrophobicities are controlled
by varying the helix length and the Leu/Ala ratio. For clarity,
we de
fi
ne a syntax for the various arti
fi
cial TMD clients to
highlight the various properties under consideration: hydro-
phobicity, length, and distribution. The generic notation is
TMD length [number of leucines], which is represented, for
example, as 18[L6] for a TMD of 18 amino acids containing six
leucines.
Our
fi
rst goal with the arti
fi
cial clients was to de
fi
ne the
minimal length of a TMD to bind to the C domain. As
described earlier in our single-point mutation capture assays,
captures of His-tagged Sgt2-TPR-C with the various TMD
clients were performed. We de
fi
ne a relative binding ef
fi
ciency
M277
M323
L327
M315
L320
I295
M290
I286
M277
I286
I295
M315
L320
M290
M323
L327
ySgt2-C
MBP-Sbh1
Sc
Sgt2
IB:MBP-Sbh1
Elution
Lysate
Pulldown: His-
Sc
Sgt2-C
Rel.Efficiency
WT
M277D
I286D M290D
I295D
M315D
L320D M323D L327D
Stdev (+⁄−)
1
0.14
0.13
0.18
0.21
0.34
0.23
0.29
0.40
0.03
0.12
0.14
0.08
0.12
0.06
0.10
0.13
A
B
hSgt2-C
MBP-Sbh1
Hs
SGTA
IB:MBP-Sbh1
Elution
Lysate
Rel. Efficiency
Pulldown: His-
Hs
SGTA-C
Stdev (+⁄−)
WT
L224D
M233D
M247D
F280D
L295D
L224D
F280D
L224D
L295D
M233D
F280D
M233D
L295D
1
0.71
0.64
0.68
0.69
0.86
0.21
0.16
0.59
0.44
0.12
0.10
0.08
0.08
0.04
0.05
0.04
0.08
0.08
L295
F280
M247
M233
L224
- 45
- 50
- 50
- 45
- 14.4
- 21.5
(kDa)
(kDa)
Figure 6. Effects on client binding of charge mutations to the putative hydrophobic groove of Sgt2-C
cons
.
For these experiments, individual point
mutations are introduced into Sgt2-C and tested for their ability to capture Sbh1 quanti
fi
ed as in
Figure 2
D
.
A
, for ySgt2-C, a schematic and cartoon model
are provided highlighting the helices and sites of individual point mutants both color-ramped for direct comparison. For the cartoon, the docked TMD
is
shown in purple. Binding of MBP-Sbh1 to His-tagged ySgt2-C and mutants were examined as in
Figure 2
E
. Lanes for mutated residues are labeled in the
same color as the schematic.
B
, same analysis as in
A
for hSgt2-C. In addition, double point mutants are included. Each capture assay was repeated three
times.
Characterization of the client-binding domain of Sgt2
8
J. Biol. Chem.
(2021) 296 100441
as the ratio of captured TMD client by a Sgt2-TPR-C
normalized to the ratio of a captured WT TA client by Sgt2-
TPR-C. In this case we replaced the TMD in our arti
fi
cial
clients with the native TMD of Bos1 (Bos1
TMD
). The arti
fi
cial
client 18[L13] shows a comparable binding ef
fi
ciency to Sgt2-
TPR-C as that of Bos1
TMD
(
Fig. 7
B
). From the helical wheel
diagram of the TMD for Bos1, we noted that the hydrophobic
residues favored one face of the helix. We explored this
“
hy-
drophobic face
”
by using model clients that maintained this
orientation while shortening the length and maintaining the
average hydrophobicity of 18[L13] (
Fig. 7
B
). Shorter helices of
14 or 11 residues, 14[L10] and 11[L8], also bound with similar
af
fi
nity to Bos1. Helices shorter than 11 residues, 9[L6] and 7
[L5], were not able to bind Sgt2-TPR-C (
Fig. 7
B
), establishing a
minimal length of 11 residues for the helix, consistent with the
dimensions of the groove predicted from the structural model
(
Fig. 3
).
Since a detected binding event occurs with TMDs of at least
11 amino acids, we decided to probe this limitation further.
The dependency of client hydrophobicity was tested by
measuring complex formation of Sgt2-TPR-C and arti
fi
cial
TMD clients containing an 11 amino acid TMD with
increasing number of leucines (11[Lx]). As shown in
Figure 7
C
, increasing the number of leucines monotonically
enhances complex formation, echoing previous results (
56
).
hSgt2-TPR-C binds to a wider spectrum of hydrophobic cli-
ents than ySgt2-TPR-C, which could mean it has a more
permissive hydrophobic binding groove, also re
fl
ected in the
milder impact of Ala replacement and Asp mutations in hSgt2-
TPR-C to TMD client binding (
Figs. 2
F
and
6
B
).
Sgt2-C preferentially binds to TMDs with a hydrophobic face
Next, we address the properties within the TMD of clients
responsible for Sgt2 binding. In the case of ySgt2, it has been
suggested that the cochaperone binds to TMDs based on hy-
drophobicity and helical propensity (
56
). In our system, our
arti
fi
cial TMDs consist of only alanines and leucines, which
have high helical propensities (
57
), and despite keeping the
helical propensity constant and in a range that favors Sgt2
binding, there is still variation in binding ef
fi
ciency. For the
Maximally Hydrophobic Face
11
11
11
11
11
45678
Binding Affinity (vs. Bos1)
TMD
Length
Hyd.
Residues
0.0
0.2
0.4
0.6
0.8
1.0
ySgt2
hSgt2
Binding Affinity (vs. Bos1)
ySgt2
hSgt2
7[L5]
9[L6]
11[L8]
14[L10]
18[L13]
BOS1
0.0
0.2
0.4
0.6
0.8
1.0
TMD Length
B
C
TMD
Length
Hyd.
Residues
11
11
14
18
566 6
Binding Affinity (vs. Bos1)
Distributed
Clustered
Distributed
Clustered
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
ySgt2
hSgt2
Distributed
Clustered
cMyc-tag
BRIL
Linker TMD (Leu/Ala)
A
D
Figure 7. Minimal requirements for client recognition by Sgt2.
A
, schematic of model clients. From capture assays, quanti
fi
cation of complex formation
in the eluate is calculated and normalized to that of complexes with Bos1
TMD
, here de
fi
ned as relative binding ef
fi
ciency.
B
, complex formation of ySgt2
(
blue
) and hSgt2 (
orange
) with the TA client Bos1
TMD
and several arti
fi
cial clients noted x[Ly], where x denotes the length of the TMD and y denotes the
number of leucines in the TMD. The helical wheel diagrams of the TMD of clients here and for subsequent panels with leucines colored in
dark orange
,
alanines colored in
pale orange
, and tryptophans colored in
gray
. Each assay was performed four times except for ySgt2-Bos1 and hSgt2-9[L6], which were
performed three times.
C
, complex formation of ySgt2-TPR-C and hSgt2-TPR-C with arti
fi
cial clients with TMDs of length 11 and increasing numbers of
leucine. Capture assays were repeated either two or three times.
D
, comparison of complex formation of ySgt2-TPR-C and hSgt2-TPR-C with arti
fi
cial clients
of the same lengths and hydrophobicities but differences in the distribution of leucines,
i.e.
, clustered (
solid line
)
versus
distributed (
dotted line
). Each assay
was performed either three or four times.
Characterization of the client-binding domain of Sgt2
J. Biol. Chem.
(2021) 296 100441
9
most part, varying the hydrophobicity of an arti
fi
cial TMD
client acts as expected, the more hydrophobic TMDs bind
more ef
fi
ciently to Sgt2-TPR-C (
Fig. 7
C
). Our C
cons
model
suggests that the hydrophobic groove of ySgt2-C protects a
TMD with highly hydrophobic residues clustered to one side
(see
Fig. 3
B
). To test this, various TMD pairs with the same
hydrophobicity, but different distributions of hydrophobic
residues, demonstrate that TMD clients with clustered leu-
cines have a higher relative binding ef
fi
ciency than those with a
more uniform distribution (
Fig. 7
D
). Helical wheel diagrams
demonstrate the distribution of hydrophobic residues along
the helix (
e.g.
, bottom
Fig. 7
D
). The clustered leucines in the
TMDs create a hydrophobic face, which potentially interacts
with the hydrophobic groove formed by the Sgt2-C
cons
region,
corresponding to the model in
Figure 3
B
.
Discussion
Sgt2, the most upstream component of the GET pathway,
plays a critical role in the targeting of TA IMPs to their correct
membranes along with other roles in maintaining cellular
homeostasis. Its importance as the
fi
rst con
fi
rmed selection
step of ER
versus
mitochondrial (
56
) destined TA IMPs ne-
cessitates a molecular model for client binding. Previous work
demonstrated a role for the C domain of Sgt2 to bind to hy-
drophobic clients, yet the exact binding domain remained to
be determined. Through the combined use of biochemistry,
bioinformatics, and computational modeling, we conclusively
identify the minimal client-binding domain of Sgt2 and pref-
erences in client binding. Here we present a validated struc-
tural model of the Sgt2 C domain as a methionine-rich helical
hand for grasping a hydrophobic helix and to provide a
mechanistic explanation for binding a TMD of at least 11
hydrophobic residues.
Identifying the C domain of Sgt2 as containing an STI1
places Sgt2 into a larger context of conserved cochaperones
(
Fig. 8
A
). In the cochaperone family, the STI1 domains
predominantly follow HSP-binding TPR domains connected
by a
fl
exible linker, reminiscent of the domain architecture
of Sgt2. As noted above, for STI1 these domains are critical
for coordinated hand-off between Hsp70 and Hsp90 ho-
mologs (
58
) as well as coordinating the simultaneous
binding of two HSPs. Both Sgt2 and the cochaperone Hip
coordinate pairs of TPR and STI1 domains by forming
stable dimers
via
their N-terminal dimerization domains
(
59
). With evidence for a direct role of the carboxylate
clamp in the TPR domain of Sgt2 for TA client binding
now clear (
21
), one can speculate that the two TPR domains
may facilitate TA client entry into various pathways that use
multiple HSPs.
Computational modeling reveals that a conserved region,
suf
fi
cient for client binding, forms a
fi
ve-alpha-helical hand,
which is reminiscent of other proteins involved in membrane
protein targeting. Like Sgt2, the signal recognition particle
(SRP) contains a methionine-rich domain that binds signal
sequences and TMDs. While the helical order is inverted,
again
fi
ve amphipathic helices form a hydrophobic groove that
cradles the client signal peptide (
60
)(
Fig. 8
B
). Here once more,
the domain has been observed to be
fl
exible in the absence of
client (
61
,
62
) and, in the resting state, occupied by a region
that includes a helix, which must be displaced (
60
). Another
helical-hand example recently shown to be involved in TA
IMP targeting is calmodulin where a crystal structure reveals
two helical hands coordinating to clasp a TMD at either end
(
Fig. 8
B
)(
63
). Considering an average TMD of 18 to 20 amino
acids (to span a
40 Å bilayer), each half of calmodulin in-
teracts with about ten amino acids. This is in close corre-
spondence to the demonstrated minimal 11 amino acids for a
TMD client to bind to the monomeric Sgt2-TPR-C. In the
context of the full-length Sgt2, one can speculate that the Sgt2
dimer may utilize both C domains to bind to a full TMD,
similar to calmodulin. Cooperation of the two Sgt2 C domains
in client binding could elicit conformational changes in the
complex that could be recognized by downstream factors, such
as additional interactions that increase the af
fi
nity to Get5/
Ubl4A.
Intriguingly, Sgt2-TPR-C preferentially binds to arti
fi
cial
clients with clustered leucines. The hydrophobic groove pre-
sented in the computational model provides an attractive
explanation for this preference. In order to bind to the hy-
drophobic groove, a client buries a portion of its TMD in the
groove leaving the other face exposed. Clustering the most
hydrophobic residues contributes to the hydrophobic effect
driving binding ef
fi
ciency and protecting them from the
aqueous environment. Indeed, when focusing on Sgt2
’
s role in
TA IMP targeting, GET pathway substrates have been sug-
gested to be more hydrophobic TMDs than Endoplasmic Re-
ticulum Membrane Complex substrates (
64
). Of these, for the
most hydrophobic substrates, such as Bos1, residues on both
sides of the TMD could be protected by a pair of C domains.
Alternatively, the unstructured N-terminal loop through H0
could act as a lid surrounding the circumference of the client
’
s
TMD. Unstructured regions participating in substrate binding
as well as capping a hydrophobic groove have been suggested
in the context of other domains,
e.g.
, with Get3 (
4
). The role
for this clustering of hydrophobic residues in client recogni-
tion and targeting merits further investigation.
What is the bene
fi
t of the
fl
exible helical-hand structure for
hydrophobic helix binding? While it remains an open question,
it is notable that evolution has settled on similar simple so-
lutions to the complex problem of speci
fi
c but temporary
binding of hydrophobic helices. For all of the domains
mentioned above, the
fl
exible helical hands provide an
extensive hydrophobic surface to capture a client-helix
—
driven by the hydrophobic effect. Typically, such extensive
interfaces are between pairs of preordered surfaces resulting in
high af
fi
nities and slow off rates. These helical hands are
required to only engage temporarily, therefore the
fl
exibility
offsets the favorable free energy of binding by charging an
additional entropic cost for ordering a
fl
exible structure in the
client-bound complex. The bene
fi
t for clients is a favorable
transfer to downstream components in the GET pathway as
seen for ySgt2 (
21
) and hSgt2 (
50
). The demonstration that
TA client transfer from hSgt2 to Get3 is twice as fast as
disassociation from hSgt2 into solution, perhaps interaction
Characterization of the client-binding domain of Sgt2
10
J. Biol. Chem.
(2021) 296 100441
with Get3 leads to conformational changes that further favor
release (
50
).
While hSgt2 and ySgt2 share many properties, there are a
number of differences between the two homologs that may
explain the different biochemical behavior. For the C
cons
do-
mains, hSgt2 appears to be more ordered in the absence of
client as the peaks in its NMR spectra are broader (
Fig. S1
,
B
and
C
). Comparing the domains at the sequence level, while
the high glutamine content in the C domain is conserved, it is
higher in hSgt2 (8.8%
versus
15.2%). The additional glutamines
are concentrated in the predicted longer H4 helix (
Fig. 1
A
).
The linker to the TPR domain is shorter compared with ySgt2
while the loop between H3 and H4 is longer. Do these dif-
ferences re
fl
ect different roles? As noted, in every case the
threshold for hydrophobicity of client binding is lower for
hSgt2 than ySgt2 (
Figs. 2
E
,
6
, and
7
) implying that the
mammalian protein is more permissive in client binding. The
two C domains have similar hydrophobicity, so this difference
in binding might be due to a lower entropic cost paid by
having the hSgt2 C domain more ordered in the absence of
client or the lack of an unstructured N-terminal loop.
The targeting of TA clients presents an intriguing and
enigmatic problem for understanding the biogenesis of IMPs.
How subtle differences in each client modulate the interplay of
hand-offs that direct these proteins to the correct membrane
remains to be understood. In this study, we focus on a central
player, Sgt2, and its client-binding domain. Through
biochemistry and computational analysis, we provide a struc-
tural model that adds more clarity to client binding both
within and outside of the GET pathway.
Experimental procedures
Plasmid constructs
MBP-Sbh1 full length,
ySgt2
95
–
346
(
ySgt2
-TPR-C),
ySgt2
222
–
346
(
ySgt2
-C),
ySgt2
260
–
327
(
ySgt2
-C
cons
),
ySgt2
266
–
327
(
ySgt2
-
Δ
H0),
hSgt2
87
–
313
(
hSgt2
-TPR-C),
hSgt2
213
–
313
(
hSgt2
-
C),
hSgt2
219
–
300
(
hSgt2
-C
cons
), and
hSgt2
228
–
300
(
hSgt2
-
Δ
H0)
Sti1/Hop
,
Sti1
ySgt2
,
hSgt2
STI1
N-dimerization
Hip
A
TPR Motif HSP70/90 binding
Client Binding
Dimerization
127
70
TPR
102
203
267
331
346
184
223
348 359
461 484 538
114
215
311 365
543
369
-4.5
(Arg)
4.5
(Ile)
Kyte & Doolittle
Ca
2+
/peptide binding
Hs
CALM2
Hs
PPP3CA
Co-translational
targeting
Oc
SRP54-M
Hs
TR-TMD
Co-chaperone
Sc
Sti1-DP1
TA-protein
targeting
ySgt2-C
cons
Sc
Sbh1-TMD
B
Figure 8. Various domain structures of STI1 and other helical-hand-containing proteins.
A
, the domain architectures of proteins with an STI1 domain
were obtained initially from InterPro (
87
) and then adjusted as discussed in the text. Each domain within a protein is colored relative to the key.
B
, structural
comparison of various hydrophobic-binding helical-hand protein complexes. For each
fi
gure only relevant domains are included.
Upper row
, color-ramped
cartoon representation with bound helices in
purple
.
Lower row
, accessible surface of each protein colored by hydrophobicity again with docked helical
clients in
purple
. In order, the predicted complex of ySgt2-C
cons
and
Sc
Sbh2-TMD, DP1 domain from yeast Sti1 with N terminus containing H0 in
gray
(
Sc
Sti1-DP1)(PDBID: 2LLV), human calmodulin (
Hs
CALM2) bound to a hydrophobic domain of calcineurin (
Hs
PPP3CA) (PDBID: 2JZI), and M domain of SRP54
from
Oryctolagus cuniculus
(
Oc
SRP54-M) and the signal sequence of human transferrin receptor (
Hs
TR-TMD) (PDBID: 3JAJ).
Characterization of the client-binding domain of Sgt2
J. Biol. Chem.
(2021) 296 100441
11