SCIENCE ADVANCES | RESEARCH ARTICLESTRUCTURAL BIOLOGY

Mechanistic insights into intramembrane proteolysis by
E. coli site-2 protease homolog RseP

Yuki Imaizumi1†, Kazunori Takanuki1†, Takuya Miyake2†, Mizuki Takemoto3, Kunio Hirata4,
Mika Hirose5, Rika Oi1, Tatsuya Kobayashi2, Kenichi Miyoshi1, Rie Aruga1, Tatsuhiko Yokoyama2,
Shizuka Katagiri1, Hiroaki Matsuura4, Kenji Iwasaki6, Takayuki Kato5, Mika K. Kaneko7,
Yukinari Kato7,8, Michiko Tajiri1, Satoko Akashi1, Osamu Nureki3, Yohei Hizukuri2*,
Yoshinori Akiyama2*, Terukazu Nogi1*

Copyright © 2022
The Authors, somerights reserved;
exclusive licensee
American Association
for the Advancementof Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
NonCommercial
License 4.0 (CC BY-NC).

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates toregulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-boundforms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification andcross-linking experiments indicated that the RseP domains surrounding the active center undergo conformationalchanges to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrateentry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays
also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane  sheetof RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of
intramembrane proteases that cleave transmembrane segments.
INTRODUCTION

1

Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho,
Tsurumi-ku, Yokohama 230-0045, Japan. 2Institute for Life and Medical Sciences,
Kyoto University, 53 Shogoinkawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
3
Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan. 4Life Science Research Infrastructure Group, RIKEN SPring-8 Center,
1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan. 5Institute for Protein
Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. 6Life
Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA),
University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan. 7Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Sendai, Miyagi 980-8575, Japan. 8Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Sendai,
Miyagi 980-8575, Japan.
*Corresponding author. Email: nogi@yokohama-cu.ac.jp (T.N.); yhizukur@infront.
kyoto-u.ac.jp (Y.H.); yakiyama@infront.kyoto-u.ac.jp (Y.A.)†These authors contributed equally to this work.
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

(group I) as human S2P (Fig. 1, A and B) (1, 14, 15). RseP is also
involved in the second step of sequential cleavage of type II membrane proteins for signal transduction. RseP cleaves the TM segmentof anti–sigma factor RseA in the extracytoplasmic stress response
after the periplasmic region of RseA is cleaved off by the membrane-­anchored protease DegS (Fig. 1D) (16–18). In RseP, two tandemly
arranged periplasmic PDZ domains (PDZ tandem) were proposed
to serve as a size exclusion filter to sterically hinder active site entry
by any substrate having a bulky periplasmic domain (19–21). This
substrate discrimination by size exclusion was also proposed for thehuman S2P, which has an extracytoplasmic PDZ domain (22). Besides, S2Ps are presumed to have a  sheet in the proximity of theactive center in the TM domain commonly (23, 24). RseP is also
predicted to have two intramembrane  hairpins (fig. S1), whichwere shown to bind the substrate near the bond that is cleaved andto contribute to substrate discrimination (24, 25). However, the absence of structural data hampers understanding of how site-1–cleaved
substrates pass through the size exclusion filter to access the active
center and of how the substrate TM segments exactly bind with theintramembrane  sheet of S2Ps. Because the active center of S2Ps ispredicted to be located within the hydrophobic milieu of the lipid
bilayer, it must both form a hydrophilic compartment around thecatalytic zinc for efficient hydrolysis and accommodate hydrophobic
segments of the substrate TM domain. Concerning the S2P family,
a crystal structure including the active center is available for the TM
domain of the archaeon Methanocaldococcus jannaschii S2P homolog(MjS2P) (26). However, MjS2P belongs to the group III subfamilyof S2P and does not have PDZ domains in the extracytoplasmic region (Fig. 1C) (15). Furthermore, MjS2P was proposed to regulate
substrate entry by using the TM helices flanking the catalytic core
TM domain as a gate, but those helices are not present in group I
S2Ps such as EcRseP and human S2P. Therefore, further structural
analysis, especially on the group I S2Ps having extracytoplasmic
PDZ domains, is essential for understanding the mechanism of
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Intramembrane proteolysis—hydrolysis of a peptide bond within thelipid bilayer—is implicated in a variety of cellular processes throughout all three domains of life, including signal transduction and membrane protein homeostasis (1–3). In humans, deregulation of thiscleavage leads to diseases such as Alzheimer’s disease (4, 5), while
signal transduction through intramembrane proteolysis is associatedwith pathogenic infections (6, 7). The substrate cleavage is catalyzed
by four distinct families of intramembrane proteases, each classified
on the basis of catalytic mechanism: the zinc metalloprotease site-2
protease (S2P), the aspartic protease presenilin/signal peptide peptidase (SPP), the serine protease Rhomboid, and the glutamic proteaseRas-converting enzyme 1 (Rce1) (8, 9), among which S2P, presenilin/
SPP, and Rhomboid cleave transmembrane (TM) segments of substrates. Eukaryotic S2Ps, including human S2P, have been identified
in signal transduction for lipid metabolism (10–12) and endoplasmic
reticulum stress responses (13), in which they perform the intramembrane proteolysis of transcription factor precursors after the extracytoplasmic cleavage of the substrates by site-1 protease (S1P). RseP from
Escherichia coli is an S2P homolog classified in the same subfamily

SCIENCE ADVANCES | RESEARCH ARTICLE
A

B

during substrate accommodation and cleavage. The results provide
crucial insights into the mechanism of the intramembrane proteolysis not only for S2Ps but also for the other families of intramembrane proteases that cleave the TM segments of substrates.

C
Cys-rich
PDZ

PDZ-N

Zn

TM5-C

TM4-C

Zn

N

EcRseP/KkRseP(group I)

D

TM6

TM5

TM3

TM2

Zn
TM3-C

TM4-N

TM1

TM6

TM4

TM3

TM1

TM4

TM3-N

TM2

TM1

C
TM5-N

N

C

TM2

PDZ-C
N

CBS

HsS2P
(group I)C

MjS2P(group III)

Extracytoplasmic stress

Extracellular
space
Outer
membraneOMP(native)N

OMP(unfolded)

PeriplasmCC

PDZ tandem

RseB

DegS

C*

PDZ-C

PDZ-N

RseA

RseA
Site-1 cleavage by DegS

N

σE

TM4

σE

Zn

Inner
membrane

MRE

RseP

Cytoplasm

Site-2 cleavage by RseP

sequential cleavage and substrate accommodation in the intramembrane proteolysis by S2Ps.
Here, we began by performing x-ray crystallographic analysis on
EcRseP and its ortholog to produce the first atomic models for thegroup I subfamily of S2P. Specifically, we have elucidated their three-­dimensional structures in complex with a peptide-mimetic inhibitor, which aided the design of mutational analyses to examine thebinding mode of substrate TM segments. Furthermore, the observed
structural differences between EcRseP and the ortholog prompted
us to examine the possibility of domain rearrangement in EcRseP
24 August 2022

Overall structure of EcRseP
The final model of EcRseP (M1 to F447) is full length except for thethree C-terminal residues and recombinant tag (Fig. 2, B to E). TM1(M1 to C33) contains two zinc-coordinating His residues, H22 and H26(fig. S4A). TM3 is divided by a loop-like bulge into two segments,
TM3-N and TM3-C. TM3-C contains the third zinc-coordinating
residue, D402 (fig. S4A). Besides the zinc ion in the active center, asecond zinc ion was bound to H86 and H87 in the cytoplasmic region. However, its physiological role is currently unknown as H86A
and/or H87A mutations did not affect the proteolytic activity (fig.
S4, B to D, and table S5). EcRseP was predicted to have two intramembrane  hairpins, the C1N loop (25) and the membrane-reentrant
 loop (MRE-loop) (24), between TM1 and TM2 (fig. S1). The corresponding regions are integrated into a four-stranded  sheet(hereafter referred to as the MRE-sheet) (fig. S5, A and B). Strand
4 corresponds to the edge strand and forms one side of the substrate-­binding site where its backbone makes direct contacts with batimastat (Fig. 3). These observations are consistent with our previous
findings that proteolytic activity is reduced by introducing Pro mutations into the two strands closest to the active site as observed here,
either into the N-terminal region of C1N (R39 to F44 corresponding
to strand 1) or into the C-terminal region of the MRE-loop (G67 toV70 corresponding to strand 4) (25). Similarly, substrate cleavage wasstrongly impaired in the G43A/I61G double mutant but not by either single mutation. In the crystal structure, G43 and I61 are proximal (G43 on the C1N loop and I61G on the MRE-loop). MjS2P
also has a membrane-embedded  sheet (26), while its topology
differs in strand order. However, strand 4 is still proximal to theactive center (fig. S5, C and D), and the arrangement of the  sheet
relative to the active center is the same as for the MRE-sheet of
EcRseP. Together, TM1 to TM3 and the MRE-sheet structurallyalign with their equivalents in MjS2P.
A hydrophilic compartment for substrate binding and cleavage
within the membrane is formed with substantial structure contributions from the MRE-sheet (fig. S6). The edge strand of the MREsheet, together with the conserved TM core region, forms a compartmentthat accommodates batimastat and the catalytic zinc ion. The interior of
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Fig. 1. Domain organization of the S2P family members and involvement of
RseP in the E. coli extracytoplasmic stress response. The topology diagrams for(A) EcRseP and KkRseP of group I, (B) human S2P (HsS2P) of group I, and (C) MjS2P
of group III, respectively. The three TM helices colored orange constitute a conserved catalytic core region. The group I S2Ps have a different number of PDZ
domains. HsS2P contains a Cys-rich region inserted into the PDZ domain. The
group III S2Ps (e.g., MjS2P) have a cystathionine--synthase (CBS) domain but noPDZ domain. Crystallographic analysis of MjS2P suggests that TM1, TM5, and TM6(light magenta) serve as the substrate entry gate where the close proximity between TM1 and TM6 forms a gate-closed state (26). TM1 and TM6 in MjS2P are lessor not conserved in HsS2P and RsePs. (D) Extracytoplasmic stress causes accumulation of unfolded or denatured outer membrane proteins (OMPs) in the periplasm,
together with dissociation of RseB from RseA. DegS, the E. coli counterpart of site-1
protease (S1P), is activated by interaction with the unfolded OMP and cleaves theperiplasmic region of the anti–sigma factor RseA (site-1 cleavage). Subsequently, RseP
performs intramembrane proteolysis of the DegS-cleaved form of RseA (site-2
cleavage), which leads to the activation of E. The PDZ tandem of RseP was proposed to sterically hinder the entry of the full-length RseA complexed with RseB. In
this study, structure-based mutational and cross-linking analyses have been conducted to address the question of how RseP accommodates the site-1–cleaved
substrates using the PDZ tandem, the PDZ C-terminal (PCT) region including H1and H2 helices, TM4, and the MRE-sheet.

Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

Crystallographic analysis of EcRseP and KkRsePFor structural analysis, we purified RseP from E. coli (EcRseP) and
its ortholog from marine bacterium Kangiella koreensis (KkRseP)(fig. S1). KkRseP restored the growth deficiency of E. coli rseP mutant cells (fig. S2A). In E. coli, KkRseP cleaved an analog of its native
substrate containing the TM segment from the K. koreensis RseA
ortholog and substrate analogs for EcRseP. Mutations to the putative KkRseP active site impaired this cleavage activity (fig. S2, B and
C). Furthermore, detergent-solubilized KkRseP can cleave E. coli
RseA, although with reduced activity as compared with EcRseP(Fig. 2J). In this study, crystal structures of EcRseP and selenomethionine (SeMet)–substituted KkRseP were determined in complex
with batimastat, an inhibitor for EcRseP (Fig. 2, Table 1, andfig. S3) (27).

H1

H2

N

RESULTSSCIENCE ADVANCES | RESEARCH ARTICLE
A

1

N

33 37

TM1

72

94

122129220224TM2

MREβ306 323 350 361 374 376

PDZ-N

PDZ-C

H1

H2

415 423

TM3

450

TM4 C

PCT

B

C

PDZ pocket

PDZ-N

D

PDZ-N

PDZ-C

PCT-H2

PCT-H1

PCT-loop
C
BAT

N
TM1–3

E

Zn

TM4

PDZ-C
TM2 PCT-H1
TM3
TM1

90˚

MREβ

PCT-H2E. coli RseP (EcRseP)

TM4
PCT-loop

F

G

PDZ-N

H
PDZ-N

BAT

TM4

PDZ-C
C

PCT-loop & H2
(disorder)

PDZ pocket

PCT-H1

N

PDZ-C
Zn

BAT

TM1–3

I
90˚

MREβ

K. koreensis RseP (KkRseP)

Time
(min)

PT

20

Mock
–

EcRseP
–
+

Mock
–

TM1

TM3

TM4
(neighbor)

KkRseP
–
+

1
10
60
1
10
60
1
10
60
1
60
1440
1
60
1440
1
60
1440

J

PCT-H1

TM4

TM2

BAT
PCT-loop & H2
(disorder)

HA-RseA(FL)
Cleaved(N-ter)

15105(kDa) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Cleaved(C-ter)

Fig. 2. Crystal structures of EcRseP and KkRseP. (A) Domain organization of EcRseP. The yellow rectangle labeled with MRE indicates the MRE-sheet. The rectangles
labeled with H1 and H2 indicate PCT-H1 and PCT-H2, respectively. (B and C) Two views of the structure of full-length EcRseP. Polypeptide chains are shown as ribbon
models. Batimastat (BAT) and zinc ions (Zn) are shown as sphere models. Each domain or motif is colored as in (A). (D and E) View of the structure of full-length EcRseP
from an outside-in perspective relative to the membrane. The PDZ tandem is highlighted with a transparent surface in (D), while the PDZ tandem is removed to visualize
the PCT region and the helix bundle of the TM domain in (E). (F to I) Structure of full-length KkRseP in the same views as that of EcRseP. In (H) and (I), the TM4 segmentfrom the crystal packing neighbor is accommodated into the TM1-TM3 cleft and is shown in a gray ribbon model. (J) In vitro substrate cleavage assay with purified RseP
proteins. 35S-Met–labeled model substrate HA-RseA148 from cell-free synthesis was incubated at 37°C for the indicated periods with EcRseP (2.5 ng/l; 50 nM), KkRseP(100 ng/l; 2.0 M), or enzyme buffer only (mock) plus 5 mM zinc chelator 1,10-phenanthroline (PT+) or 5% dimethyl sulfoxide (DMSO) (PT−) as indicated. Model substrateand cleavage products (labels, right) on a bis-tris SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel were visualized using a PhosphorImager. A representative result
from three technical replicates is shown.

this compartment, which is separated from the lipid bilayer by
TM4, is hydrophilic because of the presence of several charged orpolar residues (K71, D74, R76, E78, R97, N108, S387, N394, H405,
E412, Y428, S432, and the zinc-coordinating residues) and the exposed backbone of the edge strand (fig. S6, A and B). The previous
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

crystallographic analysis of MjS2P had also suggested that boththe exposed backbone of the edge strand and the charged residues
on the TM helices coordinate water in a channel to the active site(26). The MRE-sheet also has several charged residues on the cytoplasmic side and likely excludes lipid molecules, assisting the
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TM4
(neighbor)

TM4

SCIENCE ADVANCES | RESEARCH ARTICLE
Table 1. Crystallographic data and refinement. Values in parentheses are for the highest-resolution shell. PDB, Protein Data Bank. a.s.u., asymmetric unit.
Datasets

EcRseP

KkRseP (#1)

KkRseP (#2)

P1

P1

P21

a, b, c (Å)

47.34, 56.12, 69.67

44.56, 49.78. 76.08

46.27, 40.80, 160.24

, ,  (°)

68.2, 74.6, 69.3

86.9, 79.2, 82.2

90, 91.6, 90

Data collection
Space group
Cell dimensions

1

1

1

X-ray source

No. of molecules/a.s.u.

SPring-8 BL32XU

SPring-8 BL32XU

SPring-8 BL32XU

Wavelength (Å)

1.00000.97000.970043.77–3.20 (3.31–3.20)

49.30–3.10 (3.21–3.10)

40.80–3.15 (3.26–3.15)

10,129 (1,028)

11,493 (1,130)

10,675 (1,055)

Completeness (%)

99.7 (99.8)

99.8 (99.8)

99.6 (99.8)

Redundancy

8.4 (7.8)

135.8 (122.9)

72.0 (66.3)<I/(I)>

7.5 (1.1)

20.0 (1.4)

13.8 (1.5)

Resolution limits (Å)
No. of unique reflections

Rp.i.m.

0.098 (1.347)

0.038 (1.601)

0.081 (0.914)

CC (1/2)*

0.995 (0.417)

0.999 (0.821)

0.999 (0.602)

Refinement
Resolution limits (Å)

40.91–3.10 (3.24–3.10)

40.55–3.15 (3.32–3.15)

0.2460 (0.2952)

0.2564 (0.4159)

0.2654 (0.3595)

Rfree

0.3053 (0.3320)

0.2987 (0.4787)

0.2882 (0.4023)

3,478

3,230

3,252

Protein

3,444

3,197

3,219

Zn2+

2

1

1

Batimastat

32

32

32

No. of non-H atoms

Average B factor (Å2)

94.52

92.30

91.49

Protein

94.60

92.50

91.35

Zn2+

83.44

64.45

110.74

Batimastat

87.24

73.57

105.10Bond length (Å)

0.002

0.002

0.002

Bond angle (°)

0.48

0.50

0.44

93.92

95.01

92.12

Downloaded from https://www.science.org on August 24, 2022

43.76–3.20 (3.66–3.20)

Rwork

RMSD from ideality

Ramachandran plot
Favored (%)
Outlier (%)
PDB code

0

0

0.74

7W6X

7W6Y

7W6Z

*Correlation coefficient between intensities from random half-datasets.

hydrophilic compartment to recruit water molecules into the active
center (fig. S6C). In addition, the cytoplasmic region following theedge strand may also contribute to substrate binding and cleavage.
Many S2Ps, including those from both group I and group III, containacidic residues in the region connecting the putative edge strand
and the following TM region (fig. S1). Mutations of these acidic
residues substantially reduced the proteolytic activity in the Bacillus
subtilis group III S2P homolog SpoIVFB (23). In EcRseP, most of
the acidic residues in the cytoplasmic region downstream of the MRE-­
sheet form salt bridges with the surrounding basic residues and contributeto the formation of the hydrophilic compartment while this cytoplasmic
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

region forms a small globular domain (fig. S6D). Our structural data also
support that the acidic residues in this region are involved in binding
substrate and/or recruiting water molecules to the active center.
The PDZ tandem protrudes into the periplasmic space (Fig. 2,
B and C). The two PDZ domains form a pocket-like space (PDZ
pocket) oriented toward the TM domain containing the active center(Fig. 2, B to D). As predicted in the previous study (21), the N-terminal
residues of the PDZ C-terminal (PCT) region (P323 to T350) form anamphiphilic helix (PCT-H1) at the membrane surface (Fig. 2C).
PCT-­H1 leads to a loop region (the PCT-loop: G351 to G360) containing
a short 310 helix (PCT-SH) (Fig. 2E and fig. S1). PCT-H1 and the
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20
15

10

H

(Induction: 15 min)(Induction: 15 min)

60
40
20
0
1
55-

-MBP

2

3

4

5

6

7

8

4250-

TM1

*

40-

-RseP

TM2

n=3

80

Uncleaved
Cleaved
EcRseP

TM3-C

E23
Y69

60
40

L390F

20

R² = 0.8982

0
0

WT
M439F

20 40 60 80 100
Cleavage efficiency (%)

2

3

4

5

6

*

Uncleaved
Cleaved
EcRseP

SecB

100(%, @3.125 µM)

L435F
L438F

0
1
55-

(EcRseP)

L438F

M439F

L435F

N394F

I19F

L390F

I19F

80

20

(kDa)

BAT resistance

N394

N394F

100

40

3020-

I

120

(%, @3.125 µM)

F44F
BAT resistance

TM3-N

L66

WT

M439

60

40-

SecB

E23Q

(EcRseP)-SecB (kDa)

n=3

80

4250-

3020-

TM4

100

N394S

Edge
strand100

HA-MBP-RseA(LY1)148
Cleavage efficiency (%)

Cleavage efficiency (%)

#4

#1

#3

#2

B

L435

N394D

Batimastat (µM)

N394D

E

TM3-C

L438

N394S

0

HA-MBP-RseA(LY1)148

C

N394C

20

Batimastat (µM)

Zn

MREβ

N394F

40

WT

E23

5

0

0

WT

60

15

40

80

10

60

N394C

Y69

80

n=3

5

N394

WT
I19F
L390F
N394F
L435F
L438F
M439F

100

E23Q

I19

100

0

TM2

L390
F44

BAT resistance

120

n=3

Relative cleavage ratio(BAT 0 µM = 100%)

L66

G

BAT resistance

120

N394F

D

TM3-N

TM1

Relative cleavage ratio(BAT 0 µM = 100%)

A

N394F

80

N394C
N394S

60
40

N394D
WT

20

R² = 0.8153

0020 40 60 80 100
Cleavage efficiency (%)

PCT-loop make direct contacts with PDZ-N. The present structural
analysis also showed that the C-terminal residues of PCT (P361 toL374) also form a helix (PCT-H2), which makes a sharp turn at the
C-terminal end (G375) and leads to TM3-N (Fig. 2E). PCT-H2 isaccommodated into a cleft formed between TM1 and TM3-N and islocated just above the batimastat bound to the active center. In
EcRseP, TM4 (V423 to F447) interacts with the PCT-loop and
PCT-H2 at the periplasmic side and covers the batimastat.
Binding mode of batimastat
The peptide-mimetic batimastat adopts an extended conformationand is flanked by the edge strand of the MRE-sheet and TM3-N in
EcRseP (Fig. 3 and fig. S7A). The main chain of batimastat forms
hydrogen bonds with the main chain of L66 on the edge strand andwith the side chain of N394 on TM3. For the side chains of batimastat, the isobutyl group is oriented toward I19 in TM1 and L390 in
TM3-N, while the thienyl group is close to both of the residues thatwere reported to interact with substrates, F44 of the conserved GFG
motif (25) and Y69 on the edge strand of the MRE-sheet (24). In
addition, the phenyl group of batimastat forms van der Waals interactions with L435, L438, and M439 on TM4 (Fig. 3C).
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

On the basis of the structural data, we examined whether side
chains could also affect inhibitor (and thereby substrate) accommodation in addition to the backbone interactions with the edge strand.
I19N and I19F mutations to TM1 have been reported to reduce thesensitivity of EcRseP to batimastat (27). We further introduced Phe
mutations to the residues interacting with batimastat on TM3 andTM4 (L390 and N394 on TM3 and L435, L438, and M439 on TM4)
and examined their effects on batimastat sensitivity. To eliminate theinfluence of the C-terminal tags on this analysis, we introduced thesemutations into a tagless EcRseP construct (Fig. 3D and fig. S7, B and C).
In the wild type (WT), addition of 3.125 M batimastat reduced therelative cleavage ratio of the substrate to 40%. At the same batimastat
concentration with N394F, the relative cleavage ratio was only reduced to 90%. I19F also showed increased resistance to batimastat
as reported (27). Furthermore, batimastat resistance seems to correlate negatively with the intrinsic proteolytic activity of the mutantsin the absence of batimastat, particularly with a shorter induction of
EcRseP (Fig. 3, E and F). The N394F mutation with the strongest
impact on batimastat resistance also reduced the proteolytic activity
to a greater extent as compared to other mutations. Next, we
introduced N394C, N394S, and N394D mutations to evaluate the
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Fig. 3. Binding mode of batimastat to EcRseP and dependence on residue N394. (A) Close-up view of the batimastat-binding site. Batimastat and the residues in direct contact with batimastat are shown as stick models. The four strands constituting the MRE-sheet are shown in different colors. TM4 is omitted to visualize the bindingsite. (B) Topology diagram of the MRE-sheet. (C) Interaction between TM4 and batimastat. (D and G) In vivo batimastat sensitivity assay. E. coli YH2902 cells harboring
one plasmid for HA-MBP-RseA(LY1)148 (pYH124) and one for tagless EcRseP (pYH825) or its variants were first treated with 0, 12.5, or 3.125 M batimastat at 30°C for
10 min and then induced with 1 mM isopropyl--d-thiogalactopyranoside (IPTG) for 1 hour. The relative cleavage ratio was determined by quantitating the signal from
immunoblots and comparing the value with that for the 0 mM BAT condition. Points and error bars represent the means ± SD from three biological replicates (see fig. S7
for the raw data). (E and H) In vivo substrate cleavage assay with short induction. YH2902 cells carrying two plasmids as in (D) and (G) were incubated with 1 mM IPTG for
15 min. The cleavage efficiency was determined as described in Materials and Methods. SecB serves as a loading control. An asterisk indicates endogenous maltose-binding
protein (MBP). Bar plots and error bars represent the means ± SD from three biological replicates. (F and I) Scatterplots of the BAT resistances of the RseP mutants [relative
cleavage ratio under 3.125 M BAT concentration shown in (D) or (G)] against the cleavage efficiencies of the model substrate [shown in (E) or (H)].

SCIENCE ADVANCES | RESEARCH ARTICLE
possibility that the reduced activity in N394F is ascribed to lower
accumulation resulting from fold destabilization. We observed that
all three mutants accumulated in E. coli but still showed substantial
increase both in batimastat resistance and in reduction of proteolytic
activity (Fig. 3, G to I, and fig. S7, D and E). In particular, the reduction of proteolytic activity for the isosteric mutation N394D indicates that hydrogen bonding via the amide group of the N394 side
chain is critical for the cleavage. N394 may clamp a bound substrateat the active center as it interacted with the backbone of batimastat
at the opposite side of the MRE-sheet. In total, it is likely that thebinding mode of batimastat partly reflects that of native RseP substrates. The substrate segments to be cleaved are thought to be extended by the strand addition, as proposed in our previous work(24), and shielded from the hydrophobic milieu of the lipid bilayer
by the surrounding TM helices of RseP.

Arrangement of the PDZ tandem in EcRseP and KkRseP
on the cell membrane
To explore the possibility of domain rearrangement, we first examined
the arrangement of the PDZ tandem in EcRseP and KkRseP in thespheroplast (i.e., on the cell membrane) using a methoxypolyethylene
glycol 5000 maleimide (mal-PEG) accessibility assay. For EcRseP,
we introduced four single-Cys mutations to the PDZ tandem in thetagless construct to eliminate the impact of the C-terminal tags on
the conformation of the PDZ tandem. Consequently, we observedthat the Cys residue in A136C (located outside the PDZ pocket) was
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

Importance of D446 on TM4 for substrate cleavage
We next explored the role of TM4 in substrate cleavage. Although
TM4 is less conserved compared to the other three TM regions
within the S2P family (15) (Fig. 5A), the binding mode of batimastat
in EcRseP suggests that TM4 contributes to the formation of thehydrophilic compartment around the active site (fig. S6, A and B).
Hence, we prepared two EcRseP mutants, TM4 lacking the entire
TM4 and C-terminal tail region (F426 to the C terminus) and CTaillacking only the C-terminal tail region (D446 to the C terminus), andassessed the effect of the mutations on the cleavage. We observedthat both TM4 and CTail did not complement the EcRseP deficiency (Fig. 5B). Furthermore, both mutants virtually lost proteolytic activity, although CTail exhibited residual activity (Fig. 5C).
We next mutated conserved residues on TM4 to alanine (for G431,
M439, D446, and R449) or serine (for A442) (Fig. 5, A and D, andfig. S12) to examine their contributions to the cleavage. We foundthat only the mutation to the highly conserved D446 (fig. S1) impaired complementation and proteolytic activity. As the carboxyl
side chain of D446 is positioned for interaction with the electropositive N-terminal end of the helix dipole on PCT-H2 in the crystal
structure, the interaction between D446 and PCT-H2 may be critical for the maintenance of the proteolytic activity (Fig. 5E). Analysisof additional D446 mutants showed that only D446E retained proteolytic activity (Fig. 5C and fig. S13), although its complementation activity was much lower than that of the WT (Fig. 5B). Notably,
despite the absence of D446 in CTail, this mutant exhibited a substantial proteolytic activity. This may be due to the presence of theterminal carboxyl group of N445 in the proximity of the N-terminal
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Overall structure of KkRseP
KkRseP produced two crystal forms with similar crystal packing(fig. S8, A to D). The two crystal structures of KkRseP are almost
identical with a root mean square deviation (RMSD) of 1.34 Å for
387 C atoms (fig. S8, E and F), excluding some disordered loops.
The structures of individual domains in KkRseP are similar to thosein EcRseP, including the binding mode of batimastat via the MREsheet and side chain–backbone interactions with N387 (corresponding to N394 in EcRseP) (Fig. 2, F to I, and fig. S9). However, theKkRseP structures showed substantial differences in the domain
arrangement relative to those in EcRseP. For instance, the PDZ tandem is positioned further from the PCT region, and the C-terminal
part of PCT (corresponds to the PCT-loop and H2 in EcRseP) is
disordered (Fig. 2, F, G, and I). Thus, KkRseP adopts a PDZ-open
conformation, while EcRseP is in a PDZ-closed conformation. In
addition, TM4 moves away from the domains forming the active
center to interact with the cleft between TM1 and TM3-N in the
crystal packing neighbor (Fig. 2, H and I, and fig. S10A). TM4 alsointeracts with the batimastat bound to the active center of theneighbor. The phenyl group of batimastat makes close contacts
with V428 and L429 on TM4 (fig. S9B). In addition, residual electron density was observed close to PDZ-C. This electron density ismost likely derived from the C-terminal residues of the tobacco
etch virus (TEV) protease consensus sequence in the KkRseP construct (fig. S10, A and B). As the two neighboring KkRseP moleculesin the crystal form an asymmetric dimer, it is probable that the accommodation of TM4 into the neighbor is an artifact of the crystal
packing. However, the conformational difference between EcRseP and
KkRseP raised the possibility that the PDZ tandem, the C-terminal
part of PCT, and TM4 can rearrange without disrupting the TM
core region. Such structural changes, if they occur, would likely
affect the regulation of substrate accommodation.

efficiently modified with mal-PEG (~5 kDa). In contrast, the Cys
residues in D163C, L167C, and I304C were modified only upon the
addition of detergent (Fig. 4, A to C). In the crystal structure, L167
on PDZ-N makes direct contacts with PCT-H1, while D163 is deep in
the PDZ pocket. I304 belongs to the carboxylate-­binding loop of
PDZ-C and is located inside the pocket. These results indicate that
EcRseP on the membrane adopts the PDZ-closed conformation as
in the crystal structure where the PDZ pocket is sterically hinderedby the bilayer and the PCT region. Next, we also examined the conformation of the PDZ tandem in KkRseP on the cell membrane. To
conduct the accessibility assay on KkRseP without a C-terminal tag,
we inserted an exogenous epitope, PA14 tag (28), into a  turn between K54 and H55 in the MRE-sheet for antibody labeling. We
confirmed that the resulting KkRseP(54-PA14-55) and its four single-­
Cys mutants (P136C, E163C, F167C, and I302C) accumulated in
E. coli and maintained proteolytic activity (fig. S11). If KkRseP on
the membrane adopts the PDZ-open conformation as in the crystal
structure, then all of the four Cys mutants should be modified tosome extent in the spheroplast. Nevertheless, although E163C(proximal to PCT) underwent substantial but rather low modification, F167C (proximal to PCT) and I302C (inside the PDZ pocket)
were unmodified in the absence of detergent (Fig. 4, D to F). These
results suggest that the two modification sites (F167 and I302)
were inaccessible because of the closer proximity between the PDZ
tandem and the PCT region in KkRseP on the membrane, similar towhat is observed in the crystal structure of EcRseP (PDZ-closed)
rather than that of KkRseP (PDZ-open) (Fig. 4G). Thus, the PDZopen conformation of KkRseP in the crystal may be induced by therearrangement of the PCT region or by the accommodation of TM4
from the crystal packing neighbor.

SCIENCE ADVANCES | RESEARCH ARTICLE
EcRseP

A

B
A136

I304

A136

D163

70˚
L167

I304

C
(EcRseP)

D163C–(Triton X-100)

L167C

+

(min) 0 1 5
60-

0

1

–
5 0

1

A136C

+
5

0

1

–
5 0

1

I304C

+
5

0

1

–
5

50-

*

40-

-RseP

60-

*
1

(Contrast +)
2

3

4

5

6

7

8

1

Cys-less
–
+

+
5

0

1

5

0

1

5

0

1

5

mal-PEG-labeled

50-* -Unlabeled

40-

-RseP

60-

5040(kDa)

0

60-

50-

*

40(kDa)9 10 11 12 13 14 15 16 17 18(Contrast +)19 20 21 22 23 24 25 26 27 28 29 30

KkRseP

D

E

P136

P136

F167
I302

70˚

I302
E163

F
[KkRseP(PA)]

E163C

–

(Triton X-100)

F167C

+

(min) 0 1 5
60-

0

1

–
5 0

1

P136C

+
5

0

1

–
5 0

1

+
5

0

1

–
5

0

60-

1

0

1

–
5

0

1

PDZ

+
5

0

1

5

* 50-

5040(kDa)

1

-PA
2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18

40(kDa)Crystal
Cell(Cys-less)

+
5

G

WT

I302C

mal-PEG-labeled* -Unlabeled-PA

19 20 21 22 23 24 25 26 27 28 29 30

E

F

PCT-H1

Membraneend of the helix dipole. In total, the above mutational analysis revealed a previously unrecognized importance to substrate cleavage
for TM4 and that an Asp at position 446 is most ideal for this function in the native context. One possibility is that the electrostaticinteraction between PCT-H2 and D446 is structurally important for
the formation of the hydrophilic compartment that accommodates
the substrate. Another possibility is that this interaction is impor­tant because substrate accommodation requires the coordinatedstructural rearrangement of the PCT-H2 and TM4 observed between the EcRseP and KkRseP structures.
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

Conformational changes of the PCT region and PDZ tandem
during substrate cleavage
Several lines of evidence from this study raised the possibility that
the PDZ tandem and PCT region, probably in conjunction with TM4,
undergo structural changes to accommodate the substrate into theactive center for cleavage. Our previous in vivo photocrosslinking
experiment using p-benzoyl-l-phenylalanine (pBPA) indicated thatburied residue T341 on PCT-H1 is accessible to RseA (21). We
therefore performed an intramolecular cross-linking experiment totest whether the proteolytic activity of EcRseP is affected by immobilizing the PCT region and/or the PDZ tandem (Fig. 7, A and B).
In this assay, EcRseP mutants having two Cys mutations were firstexpressed under isopropyl--d-thiogalactopyranoside (IPTG) induction, then the cells were washed, and disulfide cross-linking was
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Fig. 4. mal-PEG accessibility assay for the PDZ tandem. (A and B) Mapping
mutation sites onto the crystal structure of EcRseP. The side chains of the mutatedresidues are shown as sphere models. The residue modified without detergent
Triton X-100 (A136) is colored red, while the other residues colored blue were modified
upon addition of the detergent. (D and E) Mapping mutation sites onto the crystal
structure of KkRseP, as in (A) and (B). The residue modified slightly without thedetergent (E163) is colored magenta. (C and F) Mal-PEG accessibility assay for the
EcRseP and KkRseP Cys mutants. Spheroplasts of KK374 cells carrying a plasmid
encoding Cys-less EcRseP (pYH835), KkRseP with internal PA14 tag (pYH838), or
one of their single-Cys mutants were treated with 1 mM mal-PEG in the presenceor absence of 2% Triton X-100 at 4°C for the indicated periods. “Contrast +” indicates a signal-enhanced image. An asterisk indicates RseP derivatives modified
with a minor mal-PEG component (62). A representative result from two biologicalreplicates is shown. (G) Conformation of the KkRseP PDZ tandem. In the crystal
structure of KkRseP, the PDZ tandem adopts an open conformation (green). However,
the mal-PEG assay indicates that the KkRseP PDZ tandem on the cell membrane
adopts a relatively closed conformation (black dotted line), in which F167 (bluecircle) is expected to be closer to the PCT-H1 (light-blue column). The partial modification of E163 (magenta circle) without detergent indicates that the KkRseP PDZ
tandem is not as close to the PCT region as the EcRseP PDZ tandem both in the
crystal and on the cell membrane.

Involvement of PCT-H2 in the substrate cleavage
To examine whether the conformational rearrangement of PCT-H2 isinvolved in the substrate cleavage, we first performed Cys-scanningmutagenesis on PCT-H2 and the adjacent region (T350 to P381)
to examine the mobility of PCT-H2 by a modified version of ouraccessibility assay with 4-acetamido-4′-maleimidylstilbene-2,2′-­disulfonate (AMS) as the probe. AMS, a soluble thiol-alkylating reagent, reacts with Cys residues exposed to the aqueous milieu. We
observed that all the Cys residues on PCT-H2 were modified atcomparable levels in the spheroplast (fig. S14), although someresidues—such as P361, I364, A365, A368, G369, and A372—are
expected to be modified less frequently if PCT-H2 is fixed in thecleft between TM1 and TM3 as in the crystal structure. Hence,
PCT-H2 is thought to be mobile in EcRseP on the cell membrane.
In contrast, Y379, L380, and P381 on TM3 showed low AMS modification in the spheroplast. The low AMS accessibility of these threeresidues is consistent with burial in the hydrophobic region of thelipid bilayer, as predicted on the basis of the crystal structure.
Furthermore, we also examined the proteolytic activity of the Cys
mutants and found that only G360C and G375C reduced the proteolytic activity (Figs. 5A and 6, A and C, and fig. S15). Considering
the high conservation of the two Gly residues flanking both endsof PCT-H2, these results suggest that the proper positioning of
PCT-H2 is important for the substrate cleavage.
Subsequently, we also tested whether PCT-H2 is involved in theregulation of the sequential cleavage. We performed a LacZ reporter
assay on the Cys mutants to monitor E activation resulting from
deregulated cleavage of intact RseA without the prior site-1 cleavage by DegS. We found that L355C, L358C, and I362C deregulate
EcRseP, although the extent of deregulation was smaller than that from
L151P, a known deregulated mutant on PDZ-N (Fig. 6, B and C)(29). We also examined trypsin susceptibility of the three mutantson the cell membrane as a proxy for stability. As reported previously,
L151P showed increased trypsin susceptibility (lanes 5 to 8; fig. S16)
probably because this mutation disrupts the folding of PDZ-N (20).
In contrast, almost no degradation was observed for the othermutants or for WT RseP (fig. S16). We also observed increased conformational flexibility in the PDZ-C domain of L358C using negative-­stain electron microscopy (EM) (figs. S17 and S18), suggesting that
the L358C mutation can deregulate EcRseP by altering the arrangement of the PDZ tandem without destabilizing the individual PDZ
domains. These results indicate that the proper positioning andmobility of PCT-H2 are also important for the regulated sequential
cleavage of substrates and likely affect the positioning of TM4.

SCIENCE ADVANCES | RESEARCH ARTICLE
A
PCT-SH

TM3

PCT-H2

TM4

P0AEH1_Ec_RseP
C7R5Z1_Kk_Kkor_1905(RseP)
D0ZKX9_Se_RseP
Q8ZH59_Yp_RseP
Q9KPV9_Vc_VC_2253
P44936_Hi_RseP
Q9CJL2_Pm_PM1991
Q9HXY3_Pa_PA3649(MucP)Q87EI0_Xf_PD_0327

B
Dilution (10X ) 0

1 mM IPTG

0.02% Ara

–1 –2 –3 –4 –5

0 –1 –2 –3 –4 –5

D

vec.
WT
E23Q
TM4
CTail

PCT-H2R449
C

PCT-SH
D446

A442

BAT

A
E
K

D446 mutation

D446(Ec)N394(Ec)

M439

G431

N
F
C

F426
Zn
30°C 21 hours

C

TM4(Induction: 3 hours)

Cleavageefficiency (%)

HA-MBP-RseA148

100
80
60
40
20
0

-MBP

n=3

1

55-

2

3

4

5

6

7

8

9

10 11

42-

*

PCT-H2

S363
PCT-SH

Uncleaved
Cleaved
EcRseP

D446

20-

SecB

TM4

CTail

WT

E23Q

vec.

(EcRseP)(kDa)

A E K N F C
D446 mutation

TM4

Fig. 5. Involvement of TM4 in the substrate cleavage and dependence on residue D446. (A) Sequence alignment of the C-terminal regions of RseP orthologs from-proteobacteria. Ec, E. coli; Kk, K. koreensis; Se, Salmonella enterica serovar Typhimurium; Yp, Yersinia pestis; Vc, Vibrio cholerae; Hi, Haemophilus influenzae; Pm, Pasteurella
multocida; Pa, Pseudomonas aeruginosa; Xf, Xylella fastidiosa. Conserved (100% match) and similar (≥50% match) residues are boxed in black and gray, respectively.
(B) Complementation assay. Cultures of E. coli KK31 cells carrying plasmids for EcRseP under the lac promoter (pYH825) or its variants were spotted on L agar plates containing IPTG or l-arabinose. A representative result from three biological replicates is shown. TM4 and CTail indicate the F426amber and D446amber mutations, respectively. (C) In vivo cleavage assay with long induction. KA306 cells harboring one plasmid for HA-MBP-RseA148 (pKA65) and one for EcRseP (pYH825) or its variants weregrown for 3 hours with 1 mM IPTG and 1 mM adenosine 3’,5’-cyclic monophosphate (cAMP). The cleavage efficiency was determined as in Fig. 3E. Bar plots and error bars
represent the means ± SD from three biological replicates. (D) Conserved residues on TM4. The residues mutated to alanine or serine in fig. S12 are shown as spheres. A
disordered region containing R449 is indicated by a dotted line. (E) Specific interaction between D446 and PCT-H2. TM4, PCT-SH in the PCT-loop, and PCT-H2 are shownas stick models in salmon, magenta, and pink, respectively. D446 interacts with the electropositive N-terminal end of the PCT-H2 helix dipole where the side chain of D446
and the main-chain N─H group of S363 (each in white) form a hydrogen bond.

induced with an oxidizing agent, diamide. As a control, we performed the same procedure but replaced the diamide with a reducing agent, dithiothreitol (DTT). Last, a model substrate was inducedwith arabinose, and cleavage was monitored with an immunoblot
assay. In this cross-linking experiment, the Cys-less EcRseP (indicated as WT* in Fig. 7A) cleaved the substrate efficiently under bothoxidizing and reducing conditions, while the active site mutant E23Q
exhibited essentially no proteolytic activity under these conditions,indicating that the diamide and DTT treatments had little impact on
the proteolytic activity. We induced cross-linking between PCT-H2
and TM1 (D7C-K366C and S10C-G369C) by treatment with theoxidizing reagent diamide and then confirmed the cross-link formation by observing the oxidization-dependent mobility shift of
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

RseP on SDS–polyacrylamide gel electrophoresis (PAGE). We actually observed that cross-linking reduced the substrate cleavage
efficiency (Fig. 7A), supporting the model that PCT-H2 moves out of
the cleft between TM1 and TM3-N to accommodate the substrate.
We also observed a reduction in the substrate cleavage efficiency forthe other double-Cys mutants, but we detected no substantial mobility shift for them (lanes 9 to 16; Fig. 7A). Despite the lack of band
shift, we conclude that both the I173C (PDZ-N)/K347C (PCT-H1)
and the D205C (PDZ-N)/S359C (PCT-loop) mutants are cross-linked
because neither could be modified at the introduced Cys residues
with a sulfhydryl-specific reagent (mal-PEG) after pretreatment withthe oxidizing reagent diamide (fig. S19). For the other two mutants,
we could not unambiguously correlate the reduction in the substrate
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50-

-RseP 40-SecBE

SCIENCE ADVANCES | RESEARCH ARTICLE
A

100

Cleavage of HA-MBP-RseA(LY1)148

TM2

PCT-H1

60

G375

40
20

TM1
PCT-loop

PCT-H2TM3-N

σE activity ∆degS

3

n=3

D446

6

2

5

1.5

TM4

4
3

1

G360

2

0.5

1
WT*
vec.
T350
G351
D352
V353
K354
L355
N356
N357
L358
S359
G360
P361
I362
S363
I364
A365
K366
G367
A368
G369
M370
T371
A372
E373
L374
G375
V376
V377
Y378
Y379
L380
P381

0

I362

8
7

2.5

(Cys at)

BAT

0

L355

L358

L151P*

0

Relative LacZ activity(WT = 1)C

80

(Cys at)

B

n=3

WT*
vec.
T350
G351
D352
V353
K354
L355
N356
N357
L358
S359
G360
P361
I362
S363
I364
A365
K366
G367
A368
G369
M370
T371
A372
E373
L374
G375
V376
V377
Y378
Y379
L380
P381

Cleavage efficiency (%)(Induction: 0.5 hours)

cleavage efficiency with the formation of an intramolecular disulfide
bond due to the diamide-dependent destabilization (D171C/S343C)
or uncertainty regarding the cross-link formation (E203C/K354C).
Collectively, the results of the cross-­linking experiments support amodel in which the PDZ tandem and the PCT region rearrange toaccommodate the substrate for cleavage. In KkRseP, the cleft between
TM1 and TM3-N accommodates the TM4 from the crystal packing
neighbor. We inferred that the bound TM4 might mimic the substrate and tested this prediction in an intermolecular photocrosslinking experiment with EcRseP and RseA. As anticipated, pBPA
introduced at the position of Y378 on the cleft (Fig. 7B) is accessible
to RseA in vivo (Fig. 7C and fig. S20), which also indicates a rearrangement of PCT-H2 during substrate accommodation.
DISCUSSION

In this study, we successfully determined the crystal structures of
EcRseP and KkRseP as the first experimental structures for S2Ps withextracytoplasmic PDZ domains (group I). The structural features not
only coincide with the previously proposed models but also provide
new insights into the substrate accommodation mechanism. Specifically, the substrate discrimination and accommodation by EcRseP
are presumed to be regulated by three processes: size exclusion, gating, and unwinding (Fig. 8).
Previous structural and mutational analyses have proposed that the
PDZ tandem serves as a size exclusion filter to regulate the sequential
cleavage of the substrates (20). It was also proposed that PCT-H1 is
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

involved in the size exclusion as an adapter (21). Furthermore, mostpoint mutations that deregulate sequential cleavage have been locatedin the PDZ-N domain (29). On the basis of trypsin susceptibility, thosederegulated mutations are presumed to cause large structural changes
or unfolding of PDZ-N (20). The present crystallographic analysis of
EcRseP has shown that the PDZ-C domain protrudes in front of
PCT-H2 to lie above the active center and thus appears to stericallyhinder entry for substrates with a bulky periplasmic domain (Fig. 2,
B to D). In addition, the PDZ-N domain makes direct contacts with
the PCT-H1 in EcRseP. Thus, the unfolding of the PDZ-N domainshould destabilize the interaction between PDZ-N and PCT-H1,
which may also perturb the geometry of size exclusion filter to deregulate its function. The Cys-scanning mutagenesis in this study further
demonstrated that the PCT-loop and PCT-H2 also regulate the sequential cleavage (Fig. 6B). The EM analysis also suggested that the
mutation to the PCT-loop region (L358C) causes deregulation
through fluctuations in the orientation of the PDZ-C (fig. S18). Theseobservations indicate that the PDZ-N domain and the PCT region
serve as a scaffold to place the PDZ-C domain in a position to performthe size exclusion function. A similar substrate discrimination mechanism was proposed for -secretase where nicastrin regulates substrateentry by steric hindrance via a bulky extracytoplasmic domain (30).
Moreover, the present study indicates that a domain rearrangement
occurs during the substrate accommodation and cleavage. On thebasis of the modification and cross-linking analyses, we infer that
the PDZ tandem, PCT-H2, and TM4 in EcRseP serve as a gate for
substrate entry. We anticipate that EcRseP on the cell membrane is
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Fig. 6. Cysteine-scanning mutagenesis analysis of the PCT-H2 and the adjacent regions. (A) Substrate cleavage with short induction. E. coli KK211 (rseA rseP) cells
harboring one plasmid for HA-MBP-RseA(LY1)148 (pYH20) and one for a variant of EcRseP(Cys-less)-His6-Myc (pTM132) were grown at 30°C in M9-based medium for
2.5 hours and further incubated for 0.5 hours with 1 mM IPTG and 5 mM cAMP. The cleavage efficiency was determined as in Fig. 3E (see fig. S15 for the raw data). Bar plots
and error bars represent the means ± SD from three biological replicates. The region of each mutation is indicated by color (magenta, pink, and gold) and label. (B) DegS-­
independent E activity of cells expressing RseP Cys mutants. Cells of rpoHP3-lacZ reporter strain AD2473 (degS rseP) harboring pSTD343 (lacI) and a plasmid for a variantof EcRseP(Cys-less)-His6-Myc (pTM101) were grown for 5 hours in L medium containing 0.1 mM IPTG and 1 mM cAMP. The measured LacZ activities are normalized as theratio to the activity for the reporter strain expressing WT RseP (WT). The bar plot shows the means ± SD from three biological replicates. Red dashed line indicates the threshold for deregulated cleavage of intact RseA by the RseP Cys mutants. The previously isolated L151P mutant (right) shows high LacZ activity characteristic of deregulation.
WT* and L151P* indicate Cys-less derivatives of WT and L151P RseP, respectively. (C) Mapping mutations on the EcRseP model. Residues where the Cys mutation impaired
the proteolytic activity are indicated with red sphere models. Residues where the Cys mutations caused deregulation are indicated with cyan sphere models.

SCIENCE ADVANCES | RESEARCH ARTICLE
A

Induction
RseP

CultureOxidationor reduction

B

Induction
substrate

D205

S359

W (0.5 hours) W (0.5 hours) W (0.5 hours)

Diamideor DTT

IPTG

Sampling

Ara

(TCA)

7C/ 10C/ 171C/ 173C/ 203C/ 205C/
EcRseP WT* E23Q* 366C 369C 343C 347C 354C 359C
Diamide + – + – + – + – + – + – + – + –DTT – + – + – + – + – + – + – + – +
5542-

-MBP

826855-

E203

K354

I173

K347

D171

S343

Y378

Uncleaved
Cleaved
**

K366

D7

G369

T341
S10

EcRseP
Red
Ox

42-

Y69

-RseP

(kDa)

Diamide

n=3

DTT

80
60

C

pBPA at Y378 None Y69
UV + – + – + –205C/359C

5040(kDa)

XL
RseA FL
RseA∆P-RseA
EcRseP-HM
1

2

3

4

-Myc
5

6

Fig. 7. Cross-linking experiments to examine the structural change during substrate cleavage. (A) In vivo proteolytic activity for EcRseP after domain immobilization
by disulfide cross-linking. AD2544 cells harboring an IPTG-inducible plasmid for EcRseP (pYH835) or its variants and an arabinose-inducible plasmid for HA-MBPRseA(LY1)148 (pTM949) were first induced with 5 mM IPTG and then treated with 5 mM diamide or 10 mM DTT. Time-course diagram is shown at the top where “w” indicates a cell wash step. After washing the cells, substrate expression and cleavage were induced with 0.02% l-arabinose for 0.5 hours. The cleavage efficiency wasdetermined as in Fig. 3E. Bar plots and error bars represent the means ± SD from three biological replicates. WT* and E23Q* indicate Cys-less derivatives of WT and E23Q
RseP, respectively. Double asterisk indicates endogenous MBP. (B) Introduction of intramolecular cross-links. Six pairs of engineered Cys are indicated by a label, and theirside chains are shown as stick models. Residues where the introduced pBPA was cross-linked with RseA are shown with sphere models. (C) In vivo photocrosslinking
between EcRseP(Y378pBPA) and RseA. KA418 (rseA+)/pEVOL-pBpF cells with pKA52 encoding RseP(E23Q)-His6-Myc (none) or pKA52 derivatives having an amber mutation at the position of Y378 or Y69 were cultured and ultraviolet (UV)–irradiated as indicated. XL indicates the cross-linked products between RseP-HM and RseA. RseA FL
and RseAP indicate the full-length and the DegS-cleaved form of RseA, respectively. RseP(Y69pBPA) with pBPA on the edge strand of the MRE-sheet was used as apositive control for cross-linking. A representative result from three biological replicates is shown.

in equilibrium between the “gate-open” and “gate-closed” conformations. The AMS modification analysis indicates that PCT-H2 ismobile on the E. coli cell membrane. After the site-1 cleavage in theextracytoplasmic region, the substrate passes through the size exclusion filter to access the TM domain of EcRseP. The substrate TM
segment is thought to enter the active center when PCT-H2 andTM4 move away together, linked by the electrostatic interaction
between the PCT-H2 backbone and conserved residue D446. On
the basis of the intramolecular cross-linking analysis, the PDZ tandem is also thought to reorient relative to the PCT region during thesubstrate accommodation, whereas the PDZ pocket is still inaccessible enough to maintain the size exclusion function. The gating
mechanisms are also presumed to exist not only in the S2P family
but also in the other intramembrane proteases such as bacterial
rhomboid GlpG (31, 32) and -secretase (33), a member of the
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24 August 2022

presenilin/SPP family. In addition, it was proposed that E. colirhomboid GlpG has an “interrogation site” for substrates in addition to a “scission site” to discriminate the substrates from nonsubstrates (34). As the cleavage efficiency in EcRseP differs dependingon the substrate, the proposed gating mechanism may also contribute to the substrate interrogation.
After the gate opening for the substrate accommodation, thesubstrate cleavage segments should bind with the edge strand via
strand addition. We anticipate that the gate returns to a relatively
closed conformation to shield the substrate TM segment from thehydrophobic milieu and to form a hydrophilic compartment aroundthe active site for hydrolysis. Furthermore, our present structural
analysis on EcRseP complexed with batimastat indicates the importance of the conserved residue N394 in the substrate binding andcleavage. We previously observed that the EcRseP(N394C) mutant
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203C/354C

15-

173C/347C

0

171C/343C

28-

10C/369C

20
7C/366C

825542-

E23Q*

40

WT*

Cleavage efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16100

SCIENCE ADVANCES | RESEARCH ARTICLE
C(1) Size exclusion
by PDZ tandem

Substrate entry after site-1 cleavage
PDZ-C

PDZ-C

PDZ-N

C*

PDZ-N

C*

ES

(Gate-closed)

Zn

N(Gate-open)

Conformational fluctuation of
PDZ tandem, PCT-H2, and TM4(2) Regulation by
substrate entry gate

ES*

D
TM4

Zn

Edge

Edge

Edge

Intactsubstrate

E

N

TM4

TM4

N

PDZ-N
H2

H2 D

D

N

PDZ-C
C*

H2

N

Substrate accommodation
for site-2 cleavage

N

Zn
Z
n

N

(Gate-closed*)(3)) Substrate unwin
unwindingby strand addition & clamping+ Batimastat

PDZ-C

PDZ-N

C*

PDZ-C
PDZ-N

C*
H2

EI

N

Edge

Zn

ESI

D
TM4

Edge

TM4

N

BAT

D

BAT

H2

N

Zn

N

(EcRseP in the crystal)Competitive inhibiton by batimastat

showed no detectable cross-linking to Cys residues engineered into
the TM segments of RseA derivatives. Thus, we inferred that N394
might not directly interact with RseA (35). However, our present
crystallographic analysis demonstrated that the N394 side chain
formed hydrogen bonds with the backbone of batimastat. Although
the structure of the EcRseP-substrate complex remains unsolved,
the present mutational analysis prompted us to propose an alternative model in which N394 directly interacts with the backbone of
the substrate TM segments to stabilize a cleavage-susceptible substrate conformation. Thus, the failure to cross-link between the
EcRseP(N394C) mutant and the RseA TM segment might be ascribed to the reduced affinity of this mutant to the substrate. We
also observed that the N394C mutation reduced the affinity for the
RseA TM segment in the coimmunoprecipitation assay (35). The
residue corresponding to EcRseP N394 in the B. subtilis homolog
SpoIVFB (N129) has been shown to be important for substratebinding and cleavage. The SpoIVFB(N129C) mutant was disulfide
cross-linked with coexpressed substrate mutants having a Cys residue
in their TM segment, albeit with low efficiency (36), indicating theproximity of N129 to the bound substrate TM segment. In addition,
the N129A mutation reduced the cross-linking efficiency of SpoIVFB
with its substrate and reduced substrate cleavage (36–38), supportingthe conclusion that N129 also stabilizes the bound substrate. Because
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24 August 2022

SpoIVFB is a group III S2P, this interaction may be common acrossdiverse S2Ps. Furthermore, clamp-like interactions with unwound
substrates appear to exist across the intramembrane proteases. The
cryo-EM analysis of -secretase classified as an aspartic protease
showed that substrate binding induces the formation of a  sheet in
the TM region of presenilin (Fig. 9, A and B) (33, 39). The  sheetbinds with the substrate fragment via strand addition. In -secretase,
the PAL motif is located on the opposite side of the  sheet like aclamp for the substrate. For the serine protease Rhomboid family,
crystallographic analysis of E. coli GlpG in complex with substrate-­
derived inhibitors revealed that two loops connecting the TM helices(the L3 and L5 loops) sandwiched the peptide backbone of the inhibitor in an extended conformation (40). In the cocrystal structures, it appeared that L3 formed a parallel  sheet with the inhibitor,
while L5 clamped it via backbone hydrogen bonds (Fig. 9, C and D).
Furthermore, GlpG was also reported to capture a substrate peptide
via the same backbone interactions with the L3 and L5 loops (31).
Despite the difference in the catalytic mechanism, unwinding theTM helix into a strand conformation and stabilizing the bound substrate with a clamp structure seem to be common features in theintramembrane proteolysis of helical TM spans. Structure determination of substrate-bound and substrate-unbound forms of RseP or
the group I S2Ps will deepen our understanding of the substrate
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Fig. 8. Proposed mechanism for substrate accommodation and cleavage in EcRseP. The substrate accommodation by EcRseP is thought to be regulated by three
processes. (1) Size exclusion process: The PDZ tandem restricts the entry of bulky intact substrates. After the site-1 cleavage, the size-reduced substrates become accessible to the TM domain of EcRseP. (2) Gating process: The PDZ tandem, PCT-H2, and TM4 serve as a gate to regulate the substrate entry into the active center. In the gateclosed conformation (labeled the E state), the active center is covered by the PDZ tandem, PCT-H2, and TM4 and is inaccessible to the substrate TM segment. The present
study suggests that the PDZ tandem is separated from the PCT region upon substrate binding (labeled the ES state). In this gate-opening movement, PCT-H2 and TM4
are also thought to move away from the active center while maintaining an electrostatic interaction via the conserved residue D446 (indicated in orange). (3) Unwinding
process: The TM segment of the substrate is extended by the edge strand of the MRE-sheet and clamped by the conserved residue N394 (indicated with a yellow roundtriangle) to promote the cleavage (labeled the ES* state). We anticipate that the gate adopts a relatively closed conformation (labeled “Gate-closed*”) again to shield thesubstrate TM segment from the hydrophobic milieu. The crystal structure of EcRseP with batimastat (gray arrow labeled “BAT”) is thought to correspond to the EI state.
The crystal structure of KkRseP with batimastat may partly reflect the ESI state, in which TM4 of the crystal packing neighbor mimics the substrate TM segment.

SCIENCE ADVANCES | RESEARCH ARTICLE
C

A
NCT

Substrate-derived
inhibitor
space
PS1 Extracellular/lumenPeriplasm

Membrane

Membrane

Cytosol

PEN-2
Substrate

B
TM5

Cytoplasm

APH1

TM7

GlpG

D385A
TM9

D

Substrate-derived
inhibitor
L5 loopTM3

L3 loopH254
PAL motif

TM6

TM8
Substrate

TM5

TM4 S201 TM2cleavage mechanism conserved across the intramembrane proteases
and aid the development of strategies for regulating proteolytic activity in the membrane to prevent off-target or promiscuous cleavage.
MATERIALS AND METHODS

Construction of expression plasmids for structural analysis
A pUC118-based plasmid was constructed to produce EcRseP for
structural analysis. The resulting plasmid, designated as pNY1452,
carried a gene encoding the WT full-length EcRseP fused with aC-terminal tag containing the TEV consensus sequence, a His8 tag,a Myc epitope, and a PA tag (41): -GT-ENLYFQG-G-HHHHHHHH-­
I-EQKLISEEDL-GVAMPGAEDDVV. For negative-stain EM, anexpression plasmid for the L358C mutant, designated as pNY1550,
was constructed from pNY1452 by the inverse polymerase chain
reaction (PCR) method.
In parallel, we also searched for RseP orthologs suitable forstructural analysis. Genes of 14 orthologs were amplified using genomic DNA obtained from the Japan Collection of Microorganisms
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

Production and purification of full-length EcRseP
and KkRseP
EcRseP was overproduced in E. coli C43(DE3) (Lucigen). E. coli
C43(DE3) cells transformed with the expression plasmid pNY1452
were grown at 30°C to an optical density at 600 nm (OD600) of 0.7 to
0.8 in LB medium [Bacto Tryptone (10 g/liter), yeast extract (5 g/liter),and NaCl (10 g/liter); without pH adjustment] supplemented with
ampicillin (50 g/ml), followed by induction of overexpression with
0.1 mM IPTG and incubation at 30°C for an additional 4 hours.
Cells were harvested by centrifugation and lysed by sonication in 10 mM
tris-HCl (pH 7.4) with 150 mM NaCl. The cell lysates were centrifuged at 40,000g for 45 min at 4°C. Subsequently, the supernatant
was separated by ultracentrifugation at 200,000g for 90 min at 4°C. The
membrane fraction collected as precipitate was suspended in 10 mM
tris-HCl (pH 7.4) with 150 mM NaCl and was ultracentrifuged again
under the same conditions. Last, the precipitated membrane fraction was suspended in 10 mM tris-Cl (pH 7.4) with 150 mM NaCl, andthe total protein was quantified using the bicinchoninic acid assay.
The resuspended membrane fraction was diluted to adjust the protein
concentration to 10 mg/ml using bovine serum albumin as a standard.
EcRseP was solubilized by adding an equal volume of a solubilization buffer containing 40 mM tris-HCl (pH 8.5), 150 mM NaCl,and 2% sucrose monododecanoate (SM) to the suspension of themembrane fraction. After incubation at 4°C for 1 hour, the mixture
was ultracentrifuged at 210,000g for 90 min. at 4°C. The supernatant
was applied to NZ-1 antibody–conjugated Sepharose resin (anti–
PA tag), and the unbound fraction was washed out with a buffer
containing 10 mM tris-HCl (pH 8.5), 150 mM NaCl, and 0.05% SM.
EcRseP was eluted from the resin with a buffer containing 10 mM
tris-HCl (pH 8.5), 150 mM NaCl, 0.05% SM, and PA14 peptide (0.1 mg/
ml; EGGVAMPGAEDDVV). The C-terminal tag was cleaved off byadding TEV protease to the elution fraction, followed by incubation
at 20°C overnight. The reaction mixture was then applied to a
Superdex 200 10/300 GL SEC column (Cytiva). The peak fraction
containing putative monomeric EcRseP was collected and again applied to the same SEC column to remove oligomeric EcRseP andaggregated TEV protease. After the second round of SEC, monodisperse EcRseP sample was obtained with high purity (~95%).
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Fig. 9. Substrate unwinding modes in intramembrane proteases. (A and B) CryoEM structure of human -secretase in complex with the Notch fragment (PDB ID:
6IDF) (33). (A) -Secretase is composed of four subunits: presenilin 1 (PS1), nicastrin(NCT), anterior pharynx-defective 1 (APH1), and presenilin enhancer 2 (PEN-2). The
Notch fragment indicated by “Substrate” in blue is accommodated in the catalytic
subunit PS1. (B) Close-up view around the active site. In this structure, the active
site residue D385 was mutated to alanine as highlighted in magenta. The C-terminal
region of Notch is unwound by forming a hybrid  sheet with  strands (orange) in
the cytosolic loop between TM6 and TM7 of PS1. The PAL motif between TM8 andTM9, highlighted with stick models, appears to fix the Notch fragment as a clamp.
(C and D) Crystal structure of E. coli GlpG in complex with the substrate-derived
inhibitor (PDB ID: 4QO0) (40). (C) GlpG has six TM helices and accommodates the
peptide-mimetic inhibitor highlighted in blue in the cavity formed at the periplasmic side. (D) Close-up view around the active site. S201 and H254, highlighted inmagenta, form the active center. The periplasmic L3 loop between TM3 and TM4 aswell as the L5 loop between TM5 and TM6 (orange) sandwich the inhibitor where
L3 forms a parallel  sheet with the inhibitor. L5 also appears to clamp the backbone of the inhibitor.

(RIKEN, Microbe Division) and subcloned into Nde I and Xho I sitesof a modified pET-21b vector. Using the constructed plasmids, theorthologs were produced with a C-terminal tag containing the TEV
consensus sequence and a His8 tag: -LESSG-ENLYFQG-QFTS-­
HHHHHHHH. To examine the production level and dispersity,
the orthologs produced in small-scale culture were subjected to detergent screening with fluorescence-detection size exclusion chromatography (FSEC) after the C-terminal His8 tag was labeled using
a peptide-based multivalent nitrilotriacetic acid (NTA) fluorescentprobe, P3NTA. From the screen, an ortholog Kkor_1905 from
K. koreensis str. DSM 16069 (KkRseP; UniProtKB: C7R5Z1), a Gram-­
negative bacterium isolated from seawater of the tidal flat (42), wasselected as a promising candidate for structural analysis. The expression plasmid carrying the KkRseP gene without codon optimization was designated as pNY1432. The highest yield of monodisperse
KkRseP sample was obtained with an optimized preparation using
n-dodecyl--d-maltoside (DDM) supplemented with cholesterylhemisuccinate (CHS). The strains and plasmids used in the structural analysis including the procedures described below are summarized in tables S1 and S2.

SCIENCE ADVANCES | RESEARCH ARTICLE

Crystallographic analysis of full-length RsePs
Batimastat (Toronto Research Chemicals) was added to the SEC
elution fractions of EcRseP and KkRseP, respectively, at a final concentration of 300 M. Subsequently, EcRseP and KkRseP were concentrated up to 7 to 12 mg/ml by ultrafiltration and incorporated
into lipidic cubic phase (LCP) by mixing the protein solution andmonoolein (NU-CHECK-PREP Inc.) with a volume ratio of 5:8 using
two syringes attached with a coupler. Crystallization conditions weresearched and optimized by microbatch crystallization method using
Laminex sandwich plates and the MemMeso screening Kit (MolecularDimensions). Aliquots (50 or 100 nl) of the protein-monoolein
mixture were dispensed onto 96-well glass plates and overlaid with
800 nl of crystallization solution using a mosquito LCP (TTP
LabTech) or Crystal Gryphon LCP (Art Robbins Instruments). The
crystals of EcRseP used for data collection were obtained in a
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

crystallization solution containing 30% (v/v) PEG 500 dimethyl ether,
100 mM NaCl, 100 mM MgCl2, and 100 mM Hepes-Na (pH 7.0),
while those of KkRseP were generated from crystallization solutions
containing 28 to 30% (v/v) PEG 500 monomethyl ether, 100 mM
NaCl, 100 mM CaCl2, and 100 mM Hepes-Na (pH 7.0). Crystals were
harvested using MicroMounts (MiTeGen) or LithoLoops (Molecular Dimensions) and flash-frozen in liquid nitrogen.
Diffraction data from the crystals obtained from the LCP crystallization were collected at SPring-8 beamline BL32XU (43) using an
EIGER X9M detector (Dectris). Microfocused x-rays with a beam sizeof 15 m by 10 m at wavelengths of 1.0000 Å (EcRseP native data),
1.2800 Å (Zn-anomalous diffraction data), and 0.9700 Å (Se-anomalous
diffraction data), respectively, were used for both raster scan anddata collection. A dataset with a total oscillation range of 10° and
0.1° oscillations per frame was collected from each crystal under anabsorbed dose of 10 megagrays. The partial datasets collected withthe automated data collection system ZOO (44) were merged, integrated, and scaled using the KAMO system (45), which integrates
BLEND (46), XDS, and XSCALE (47, 48). Diffraction intensities
were converted to structure factors using the CCP4 suite where
5% of the unique reflections were randomly selected as a test set forthe calculation of free R factor (49).
Initial phases of the SeMet-substituted KkRseP crystal were determined by a combination of molecular replacement and single-­
wavelength anomalous diffraction methods (MR-SAD). After MR
using the structure of KkPDZ-C domain as a search model, the SeSAD phasing was performed using phenix.autosol (50). The initialmodels were manually modified and fit into the electron density
map using the program COOT (51). The updated models were refinedwith phenix.refine (52) iteratively while monitoring the stereochemistry with MolProbity (53). The structure of EcRseP was solved
by MR using the partial model of KkRseP in addition to the individual
EcPDZ-N [Protein Data Bank (PDB) code: 2ZPL] and EcPDZ-C(PDB code: 2ZPM) models (29). The PCT domain and TM4, where
the conformations were largely different from those of KkRseP, weremodeled manually. Model modification, structure refinement, andvalidation were performed similarly to those for KkRseP. Statistics
for data collection and refinement are summarized in Table 1.
Structural superposition and RMSD calculation were performed
by a pairwise alignment protocol using LSQKAB (54). Figures
for protein structures were prepared with PyMOL (The PyMOL
Molecular Graphics System, version 2.3, Schrödinger LLC).
Media for biochemical analysis
L medium [Bacto Tryptone (10 g/liter), yeast extract (5 g/liter), andNaCl (5 g/liter); pH adjusted to 7.2 by using NaOH] and M9 medium[without CaCl2; (55)] supplemented with thiamine (2 g/ml) and
0.4% glucose were used to culture E. coli cells. Ampicillin (50 g/ml),
chloramphenicol (20 g/ml), and spectinomycin (50 g/ml) were added
for selecting transformants and for growing plasmid-harboring cells.
In vivo proteolytic activity assay of EcRseP and KkRseP
and their derivatives
The in vivo proteolytic activities of EcRseP and KkRseP were analyzed essentially as described previously (24, 56). Precultured cells
were inoculated into an M9 medium supplemented with 20 g/ml
of each of the 20 amino acids, thiamine (2 g/ml), 0.4% glucose, 1 mM
IPTG, and 1 mM adenosine 3′,5′-cyclic monophosphate (cAMP)and grown at 30°C for 3 hours. The proteins were precipitated by
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To produce KkRseP, the plasmid pNY1432 was transformed into
E. coli C43(DE3) harboring pRARE2. For the native KkRseP, E. coli
cells were grown at 30°C to an OD600 of 0.6 to 0.7 in an LB medium
supplemented with ampicillin (50 g/ml) and chloramphenicol(34 g/ml), followed by induction of overexpression with 0.1 mM
IPTG and incubation at 16°C for an additional 18 hours. To produce the SeMet-substituted KkRseP, E. coli cells were first cultured
in an LB medium supplemented with ampicillin (50 g/ml) and
chloramphenicol (34 g/ml) grown at 30°C overnight. The cells
were further inoculated into M9 medium supplemented with ampicillin (50 g/ml) and chloramphenicol (34 g/ml) and grown at
30°C. At an OD600 of 0.3, a mixture of amino acids was added to themedium where the final concentrations were as follows: lysine(100 mg/ml), phenylalanine (100 mg/ml), threonine (100 mg/ml),
valine (50 mg/ml), leucine (50 mg/ml), isoleucine (50 mg/ml), andSeMet (60 mg/ml). At an OD600 of 0.7 to 0.8, 0.1 mM IPTG wasadded to induce the overproduction, followed by incubation at
16°C overnight. Harvested cells were lysed by sonication in 10 mM
tris-HCl (pH 7.4) with 150 mM NaCl, followed by centrifugation at
20,000g for 30 min at 4°C. Subsequently, the supernatant was further
separated by ultracentrifugation at 200,000g for 60 min at 4°C. The
collected membrane fraction was lastly suspended in 20 mM tris-HCl(pH 8.0), 300 mM NaCl, 15% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and the total protein concentration was
adjusted to 10 mg/ml.
KkRseP was solubilized by adding an equal volume of a solubilization buffer containing 40 mM tris-HCl (pH 8.0), 300 mM NaCl,
20 mM imidazole, 10% glycerol, 2% DDM, and 0.4% CHS to thesuspension of the membrane fraction. After incubation at 4°C for
1 hour, the mixture was ultracentrifuged at 210,000g for 30 min at
4°C. The supernatant was applied to Ni-NTA agarose resin, and theunbound fraction was washed out with a buffer containing 20 mM
tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 0.03% DDM,
0.006% CHS, and 50 mM imidazole. EcRseP was eluted with a buffer
containing 20 mM tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol,
0.03% DDM, 0.006% CHS, and 300 mM imidazole. The C-terminal
tag was cleaved off by adding TEV protease to the elution fraction,
followed by incubation at 20°C overnight. The reaction mixture wasdialyzed against a buffer containing 20 mM tris-HCl (pH 8.0), 300 mM
NaCl, 10% glycerol, 0.03% DDM, 0.006% CHS, and 10 mM imidazole
and was applied to the Ni-NTA agarose resin, and the tag-cleaved
KkRseP was collected in the unbound fraction. KkRseP was further
purified with a Superdex 200 10/300 GL SEC column (Cytiva).

SCIENCE ADVANCES | RESEARCH ARTICLE
trichloroacetic acid (TCA) treatment, washed with acetone, suspended in SDS sample buffer with 2-mercaptoethanol (2ME), andanalyzed by Laemmli SDS-PAGE and immunoblotting using an
Immobilon membrane filter (MilliporeSigma) and appropriate
antibodies. Proteins were visualized by a Lumino image analyzer
LAS-4000 mini (Cytiva) using ECL (enhanced chemiluminescence)or ECL Prime Western Blotting Detection Reagents (Cytiva). Rabbit
polyclonal anti–hemagglutinin (HA) [HA-probe (Y-11), Santa Cruz
Biotechnology], anti–maltose-binding protein (MBP) (57), anti-­EcRseP(19), and anti-SecB (21) antibodies and rat monoclonal anti-PA antibodies (58) were used for immunoblotting. Also, for detection of Histagged proteins, anti-His antibodies from the Penta-His HRP
Conjugate Kit (Qiagen) were used. In the short induction method,
precultured cells were inoculated into an L medium with 0.4% glucose and grown at 30°C for 2.5 hours. Collected cells were suspended in 100 l of L medium and preincubated at 30°C for 10 min
in an Eppendorf Thermomixer comfort (600 rpm). After the addition of 1 mM IPTG, the cells were incubated at 30°C for 15 min withshaking. Last, the proteins were precipitated by the TCA treatment
described above. Cleavage efficiencies of the substrates were calculated
according to the following equation: cleavage efficiency (%) = 100 ×(cleaved)/[(cleaved) + (full length)], where (cleaved) and (full length)
are intensities of the respective bands.

In vivo batimastat sensitivity assay
Batimastat inhibition of in vivo proteolytic activity of RseP derivatives was evaluated using a mutant strain that lacks the acrA genecoding a component of a multidrug efflux pump to increase the sensitivity to batimastat as described previously (27) with several modifications. YH2902 (rseA rseP acrA) cells harboring two plasmids
encoding an EcRseP derivative (pYH825 based) and a model substrate HA-MBP-RseA(LY1)148 (pYH124) were inoculated into L
medium with 0.4% glucose and grown at 30°C for 2.5 hours.
Collected cells were suspended in L medium and divided into three
portions. Each portion received 12.5, 3.125, or 0 M (final concentration) batimastat (MilliporeSigma, the stock solution of batimastat
was dissolved in DMSO) and preincubated at 30°C for 10 min in an
Eppendorf Thermomixer comfort (600 rpm). Samples were mixed
Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

Substituted cysteine accessibility analysis of single-cysteine
EcRseP or KkRseP mutants
A mal-PEG accessibility assay was performed with single-Cys mutants of
EcRseP or KkRseP using essentially the same method as described
previously (20, 56). KK374 cells (57) carrying a plasmid encoding
a tagless EcRseP Cys mutant or a KkRseP Cys mutant with an internal PA14 tag were cultured in L medium containing 0.4% glucose, 1 mM
IPTG, and 1 mM cAMP at 30°C for 2.5 hours and converted tospheroplasts by lysozyme/EDTA treatment as described previously(29). Spheroplast samples were treated with water or 2% Triton
X-100 at 0°C for 3 min in the presence of 5 mM MgCl2, 1 mM PMSF,
and 1 mM tris(2-carboxyethyl)phosphine (TCEP; MilliporeSigma).
Samples were mixed with an equal volume of 2× reaction buffer[60 mM tris-HCl (pH 8.1) and 2 mM mal-PEG (MilliporeSigma)]and incubated at 4°C for the indicated periods. The Cys modification reaction was stopped by adding 10% 2ME. The proteins were TCA-­
precipitated and analyzed by SDS-PAGE and immunoblotting.
Single-Cys EcRseP mutants were modified in two steps with AMS
and mal-PEG using essentially the same method as described previously (21, 56). Spheroplast samples of KK374 cells expressing asingle-Cys mutant of RseP-His6-Myc (RseP-HM) were prepared asabove and then treated with water or 1% Triton X-100 at 0°C for
30 min in the presence of 10 mM MgCl2, 1 mM PMSF, and 1 mM
TCEP. After prewarming at 24°C for 5 min, samples were treatedwith 1 mM AMS (Thermo Fisher Scientific) in the presence or absence of 1% Triton X-100 at 24°C for 5 min. Following quenching of
AMS by incubation with 62.5 mM DTT at 24°C for 18 min, the proteins were precipitated and washed with 5% TCA. Samples weresolubilized in 100 mM tris-HCl (pH 8.1) containing 1% SDS and
1 mM TCEP and treated with 5 mM mal-PEG at 37°C for 1 hourwith vigorous shaking to modify free thiols. AMS/mal-PEG–modified
proteins were analyzed by SDS-PAGE and immunoblotting. The
proportion of RseP-HM Cys mutants modified with AMS was
calculated according to the following equation: AMS modification(%) = 100 × (a − b)/a, in which a is the ratio of the mal-PEG–­
modified forms to total RseP-HM in the control sample that is
prepared without AMS treatment, and b is the ratio of the malPEG–modified forms to total RseP-HM in the AMS-treated sample.
For immunoblotting analysis of mal-PEG–labeled proteins, an
Immobilon-PSQ membrane filter (MilliporeSigma) and transfer
buffer containing 24 mM tris-base, 192 mM glycine, 10% methanol,and 0.05% SDS were used.
Complementation assay
E. coli KK31 [rseP/pKK6 (PBAD-rseP)] cells harboring a plasmid
encoding EcRseP or its derivatives under the lac promoter were grown at
30°C in an L medium containing 0.02% l-arabinose for 2 hours. The
cultures were serially diluted with saline, and 3 l of the diluted cultures was spotted on L agar plates containing 1 mM IPTG to test complementation or 0.02% l-arabinose as a control for monitoring total cellcounts. The plates were incubated at 30°C for the indicated period.
-Galactosidase activity assay
The E activity was assayed by monitoring -galactosidase (LacZ)
activity expressed from a chromosomal E-dependent lacZ reporter
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In vitro substrate cleavage assay with purified
EcRseP or KkRseP
Model substrate HA-RseA148 was synthesized using the PUREfrex
1.0 reconstituted cell-free protein synthesis kit [Gene Frontier Co.,
Japan; (59, 60)], using a template DNA (PCR-amplified from pYH18)
and standard reagents including 0.02% DDM and 35S-labeled methionine (35S-Met). EcRseP or KkRseP was purified according to thesame procedures as those for the samples for crystallization. Synthesized 35S-labeled substrate was mixed with purified EcRseP [final
concentration, 2.5 ng/l (50 nM)], KkRseP [final concentration,
100 ng/l (2.0 M)], or each of the respective protein buffers (mock)and incubated at 37°C in buffer containing 50 mM tris-HCl (pH 8.1),
2.5% glycerol, 5 M zinc acetate, 0.05% DDM, 100 mM NaCl, 10 mM
DTT, and 5 mM zinc chelator 1,10-phenanthroline (PT+) or 5% dimethyl
sulfoxide (DMSO; PT−) with shaking in an Eppendorf Thermomixer
comfort (500 rpm) for the indicated periods. Samples were thenmixed with an equal volume of 2× SDS sample buffer plus 2ME andboiled for 5 min. The proteins were separated by SDS-PAGE using
a 15% bis-tris gel and MES SDS running buffer (61) and visualizedusing a PhosphorImager BAS5000 (Cytiva).

with IPTG (final concentration, 1 mM) to induce protein expression and incubated for 1 hour with shaking. Proteins were TCA-­
precipitated and analyzed by SDS-PAGE and immunoblotting.

SCIENCE ADVANCES | RESEARCH ARTICLE
gene (rpoHP3-lacZ). Cells were grown at 30°C for 5 hours in an L
medium with 0.1 mM IPTG and 1 mM cAMP. The LacZ activity ofgrowing cells was measured using essentially the same method as
previously described (21).

using the genetic information processing software Genetyx (GENETYX
Corporation, Japan).
SUPPLEMENTARY MATERIALSSupplementary material for this article is available at https://science.org/doi/10.1126/

Site-directed in vivo photocrosslinking
sciadv.abp9011
Site-directed in vivo photocrosslinking was carried out using essentially the same method as previously described (21). Cells harboring REFERENCES AND NOTES
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Multiple alignment analysis of bacterial RseP homologs
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nih.gov/Blast.cgi) was performed, using the amino acid sequence of
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Disulfide cross-linking–mediated domain
immobilization experiment
The in vivo proteolytic activity of EcRseP after domain tethering by
disulfide cross-linking was examined as follows. AD2544 cells
harboring two independently inducible plasmids, each encoding an
RseP derivative with a pair of Cys mutations under the Plac promoter(pYH835 based) or the model substrate HA-MBP-RseA(LY1)148(pTM949) under the PBAD promoter, were used. Precultured cells
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1 mM TCEP and incubated at 30°C for 30 min to induce substrate
expression. Last, proteins were precipitated with TCA, resuspended
in SDS sample buffer without 2ME, and analyzed by SDS-PAGE
and immunoblotting.
To evaluate the efficiency of intramolecular disulfide cross-linking
for double-Cys RseP, all TCA-precipitated postreaction samples
were dissolved in 100 mM tris-HCl (pH 8.1) with 1% SDS andvigorously shaken for 30 min at room temperature. Then, the samples
were divided into two portions, treated with 0 or 1 mM mal-PEG at
37°C for 1 hour with vigorous shaking to modify free thiols, mixed
with 2× SDS sample buffer containing 2ME, and lastly analyzed by
SDS-PAGE and immunoblotting.

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Imaizumi et al., Sci. Adv. 8, eabp9011 (2022)

24 August 2022

Submitted 4 March 2022
Accepted 1 July 2022
Published 24 August 2022
10.1126/sciadv.abp9011

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Acknowledgments: We are grateful to the beamline staff of SPring-8 BL32XU (Hyogo, Japan)
and Photon Factory (Tsukuba, Japan) for providing data collection facilities and support. We
thank the National BioResource Project (NBRP)—E. coli at the National Institute of Genetics,
Japan for the KEIO strain. We also thank S. Thompson for critical reading and editing of themanuscript, J. Takagi for valuable advice in the early stage of this study, H. Takahashi andA. Kidera for valuable discussions, T. Nishizawa and K. Takeda for advice on LCP crystallization,
S. Tabata and T. Hirose for contribution to antibody-assisted EM analysis, and Y. Yamagata andS. Honna for contribution to in vitro substrate cleavage analysis. Funding: This research ispartially supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI under
grant numbers JP19687004, JP22370039, JP26291016, JP19H03170, and JP22H02561 (to T.N.);
under JP21J15841 (to T.Y.); under JP19K06562 and JP22K06142 (to Y.H.); under JP18H02404
and JP22H02571 (to Y.A.); and under JP21K19236 (to S.A.); by the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) under JP19H05774 (to S.A.); by the Platform
Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug
Discovery, Informatics, and Structural Life Science) from the Japan Agency for Medical
Research and Development (AMED) under grant number JP16am0101020 (to T.N.); by the
Platform Project for Supporting Drug Discovery and Life Science Research [Basis for
Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED under
grant numbers JP21am0101078 and JP22ama121008 (to Y.K.); by the Sumitomo Foundation,

Basic Science Research Projects (to T.N.); by the Astellas Foundation for Research on Metabolic
Disorders (to T.N.); by the grant for 2018 Research Development Fund of Yokohama City
University (to T.N.); by the Institute for Frontier Life and Medical Sciences, Kyoto University for
INFRONT Office of Director’s Research Grants Program (2020) and (2021) (to Y.H.); and by theInstitute for Fermentation, Osaka, G-2022-2-108 (to Y.H.). This work was performed, in part,
under the Cooperative Research Program (Joint Usage/Research Center program) of theInstitute for Frontier Life and Medical Sciences, Kyoto University (to T.N.); under the
Collaborative Research Program of the Institute for Protein Research, Osaka University,
CR-21-06 (to T.N.); and under the Cooperative Research Project Program of Life Science Center
for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA Center), University ofTsukuba (to T.N.). Author contributions: T.N., Y.H., and Y.A. conceived the project. T.N.
supervised the structural analysis. R.O. contributed to preparation of the full-length RseP. M.
Tak. and O.N. identified the RseP ortholog suitable for structural analysis. K.M. and S.K.
performed the crystallographic analysis of the partial fragment. Y.I. and K.T. performed the
crystallographic analysis of the full-length RsePs. K.H. and H.M. collected and analyzed thediffraction data. R.A., M.H., K.I., and T.Ka. performed the EM analysis. M.K.K. and Y.K.
contributed to the preparation of the antibody fragment. M.Taj. and S.A. performed the mass
spectrometry analysis. Y.A. and Y.H. supervised the biochemical analysis. Biochemical analyses
were performed by T.M., T.Ko., and Y.H. (in vivo) and by T.Y. (in vitro). All the authors
contributed to paper preparation. T.N., Y.A., and Y.H. wrote the original draft of themanuscript, and T.N. compiled the paper. Conceptualization: Y.H., Y.A., and T.N. Methodology:
K.H., K.I., T.Ka., Y.K., S.A., O.N., Y.H., Y.A., and T.N. Investigation: Y.I., K.T., T.M., M.Tak., K.H., M.H.,
R.O., T.Ko., K.M., R.A., T.Y., S.K., H.M., M.K.K., M.Taj., Y.H., Y.A., and T.N. Funding acquisition: Y.K.,
T.Y., S.A., Y.H., Y.A., and T.N. Project administration: K.I., T.Ka., Y.K., S.A., O.N., Y.H., Y.A., andT.N. Supervision: Y.H., Y.A., and T.N. Writing—original draft: Y.H., Y.A., and T.N. Writing—review
and editing: T.M., M.Tak., K.H., M.H., R.A., Y.K., M.Taj., Y.H., Y.A., and T.N. Competing interests:
The authors declare that they have no competing interests. Data and materials availability:
The atomic coordinates were deposited in the PDB with the accession codes as follows:
full-length EcRseP (7W6X), full-length KkRseP in the P1 crystal (7W6Y), full-length KkRseP in theP21 crystal (7W6Z), KkPDZ-C domain (7W70), and EcPDZ-C domain complexed with the 12C7
Fab (7W71). The negative-stain EM maps were deposited in the Electron Microscopy Data Bank
with accession codes as follows: WT EcRseP complexed with the 12C7 Fab (EMD-33409) and
EcRseP(L358C) mutant complexed with the 12C7 Fab (EMD-33410). All other data needed toevaluate the conclusions in the paper are present in the paper and/or the Supplementary
Materials.

Mechanistic insights into intramembrane proteolysis by E. coli site-2 proteasehomolog RseP
Yuki ImaizumiKazunori TakanukiTakuya MiyakeMizuki TakemotoKunio HirataMika HiroseRika OiTatsuya
KobayashiKenichi MiyoshiRie ArugaTatsuhiko YokoyamaShizuka KatagiriHiroaki MatsuuraKenji IwasakiTakayuki
KatoMika K. KanekoYukinari KatoMichiko TajiriSatoko AkashiOsamu NurekiYohei HizukuriYoshinori AkiyamaTerukazu
Nogi

Sci. Adv., 8 (34), eabp9011. • DOI: 10.1126/sciadv.abp9011

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