JBC Papers in Press. Published on May 4, 2006 as Manuscript M600018200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M600018200

CONTROL OF CHEMOTACTIC SIGNAL GAIN
VIA MODULATION OF A PRE-FORMED RECEPTOR ARRAY*

Hiroki Irieda1, Motohiro Homma1§, Michio Homma1, and Ikuro Kawagishi1,2
From 1Division of Biological Science, Graduate School of Science, and 2Institute forAdvanced Research, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
Running title: Bacterial chemoreceptor array
Address correspondence to: Ikuro Kawagishi, Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan, Tel, +81 52-789-2993; Fax, +81
52-789-3001; E-mail, i45406a@cc.nagoya-u.ac.jp§
Present address: Department of Life Sciences, Graduate School of Arts and Sciences, University ofTokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan.
The remarkably wide dynamic range ofthe chemotactic pathway of Escherichia coli, a
model signal transduction system, is achievedby methylation/amidation of thetransmembrane chemoreceptors, which
regulate the histidine kinase CheA in response
to extracellular stimuli. The chemoreceptors
cluster at a cell pole together with CheA andthe adaptor CheW. Several lines of evidence
have led to models that assume highcooperativity and sensitivity via collaborationof receptor dimers within a cluster. Here,using in vivo disulfide crosslinking assays, wedemonstrate a well-defined arrangement of theaspartate chemoreceptor (Tar). The
differential effects of amidation on crosslinking
at different positions indicate that amidation
alters the relative orientation of Tar dimers toeach other (presumably inducing rotational
displacements) without much affecting theconformation of the periplasmic domains.
Interestingly, the effect of aspartate on
crosslinking at any position tested was roughly
opposite to that of receptor amidation.
Furthermore, amidation attenuated the effects
of aspartate by several orders of magnitude.
These results suggest that receptor covalentmodification controls signal gain by altering
the arrangement or packing of receptor dimers
in a pre-formed cluster.
Chemotaxis of Escherichia coli is one of
the most extensively studied sensory systems,
recognizing the concentration of environmental
chemicals and migrating toward the favorite
direction (for reviews, see 1-5). All of thecomponents have been identified and extensively
studied. However, the molecular mechanisms

underlying its high sensitivity and wide dynamic
range have not been fully understood. The
chemotactic signal is transmitted from thechemoreceptors to the flagellar motor via astoichiometric His-Asp phosphorelay from thehistidine kinase CheA to the response regulator
CheY. The chemoreceptors of Escherichia coli
belong to one of the best studied transmembrane
receptor families. The receptor cytoplasmic
domain interacts with CheA and the adaptor
protein CheW (6, 7) and the resulting ternarycomplexes form a cluster at a cell pole (8-10).
Attractant binding to the Tar dimer, which isformed regardless of its ligand occupancy state(11), induces a small but critical inward
displacement of a membrane-spanning α-helix of
one subunit (12-17). This displacement isthought to trigger a structural change in thecytoplasmic domain, which then inactivates CheA.
To account for high sensitivity of the chemotaxis
system, however, it has been proposed that
attractant binding also affects neighboring
receptor dimer(s) (18-21), models which havebeen supported by several lines of evidence(21-26). Receptor clustering has also been
implicated in signal gain control by methylation(or amidation) of specific glutamate residues thatis responsible for adaptation to persisting stimuli.
Only a slight decrease in the attractant-binding
affinity (27-30) and a slight increase in the CheA
activity (28, 30) that result from receptor covalentmodification cannot account for adaptation.
Rather, receptor methylation/amidation seems tocontrol signal gain presumably through receptor
clustering (21, 23, 30). However, receptor
methylation/amidation does not drastically alter
the polar localization of the high-abundance
chemoreceptors (31-33).

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

We have already established, by using asite-directed disulfide crosslinking assay, thatreceptor dimers interact with each other in vivo
and that this interaction is modulated by attractant
binding (26). Here, we employed this technique
to ask whether receptor clustering is involved ingain control. Systematic disulfide scanning
revealed a well-defined array of receptor dimers.
Crosslinking at different positions were affecteddifferentially by receptor amidation (equivalent tomethylation), suggesting that receptor amidation
alters relative orientation of receptor dimers in thecluster rather than inducing their association or
dissociation. In any position tested, attractant
binding showed effects roughly opposite toamidation but these attractant effects wereattenuated by increasing levels of amidation.
These results suggest that receptor amidation(methylation) controls signal gain by altering thearrangement or packing of receptor dimers in a
pre-formed cluster.

harvested and suspended in SDS-loading buffer[35 mM Tris-HCl (pH 6.8), 6.7% glycerol, 1%
SDS, 0.0007% BPB] supplemented with 2.5 mM
NEM and 2.5 mM EDTA. When necessary,
2ME was added to the final concentration of
12.5%. Samples were transferred onto a
polyvinylidene difluoride membrane (Millipore
Japan, Tokyo) using a semidry blotting apparatus(Biocraft, Tokyo). The antibody raised against
the C-terminal peptide[NH2-(C)PRLRIAEQDPNWETF-COOH] of Tar(αTar-C) was prepared by Sawadi Technology Co.
(Tokyo). The HRP-linked anti-rabbit IgG
antibody (Cell Signaling Technology, Beverly,
MA, U.S.A.) was used as the second antibody.
The protein-antibody complexes were visualizedwith ECL Western-blotting detection reagents(Amersham Pharmacia Biotech).
To quantify the intensity of the Tar band,the immunoblots were scanned and the resulting
images were analyzed by using the software
ImageJ(http://rsb.info.nih.gov/ij/).
The
crosslinking efficiency (in percentage) of a given
Tar mutant protein was defined as a proportion of
the crosslinked dimers to the total amount. For
each mutant, crosslinking assays were triplicatedwith three independent transfromants and themean and standard deviation values ware
calculated.
To examine the effects of an attractant on
disulfide crosslinking, cells were grown for 3.5 h
as described above, washed twice with EDTA-free
MLM medium [10 mM potassium phosphatebuffer (pH 7.0), 10 mM DL-lactate, 0.1 mM
methionine], resuspended in EDTA-free MLM,
and divided into aliquots. MeAsp (0-10-3 M)
was added to each aliquot. The samples wereincubated for 10 min at room temperature or at
30˚C, before being treated with an oxidizing
catalyst Cu(II)(o-phenanthroline)3 (hereafterreferred to as Cu-phenanthroline) (0-200 µM)
supplemented with MeAsp (0-10-3 M) for 10 min
at room temperature or at 30˚C. To stop theoxidation reaction, 1/5 volume of prechilled stop
solution [210 mM Tris-HCl (pH 6.8), 15 mM
EDTA, 15 mM NEM] supplemented with MeAsp(0-10-3 M) was added to the samples and thensamples were put on ice. Cells were collected
and suspended in SDS-loading buffer containing
2.5 mM NEM and 2.5 mM EDTA supplemented

Experimental Procedures
Bacterial strains and plasmids—Strain HCB339(34) lacks all four chemoreceptors, whereas strain
HCB436 (35) lacks all four chemoreceptors,
CheB, and CheR. All Tar-encoding plasmids
used for the crosslinking assays were derived
from pWSK29 (36), a derivative of pSC101 thatcarries the bla gene. Site-directed mutagenesisof tar was carried out essentially as described
previously (26).
Swarm assay of chemotaxis—Swarm assays were
performed with tryptone semisolid agar (1%tryptone, 0.5% NaCl, 0.3% agar) supplemented
with 50 mg/ml ampicillin. After swarm plates
were inoculated with fresh colonies, they wereincubated at 30˚C for 10 to 20 h. In someexperiments, cell suspensions were spotted onto aplate, which was then incubated at 30˚C for 8 to 9h.
In vivo disulfide crosslinking—Disulfide
crosslinking and immunoblotting were essentiallyas described previously (26). TG broth [1%tryptone, 0.5% NaCl, 0.5% (w/v) glycerol]
supplemented with 50 mg/ml ampicillin was
inoculated at 1:30 dilution with a fresh overnight
culture of cells carrying a plasmid. Cultures
were then shaken at 30˚C. After 3.5 h, cells were2with MeAsp (0-10-3 M). When necessary, 2ME
was added. Samples were analyzed byimmunoblotting as described above.

native structure. The Cys residues in the
C-terminal half of loop 2-3 (P114C-V117C) weremore apt to be crosslinked than those in the
N-terminal half (K108C-L113C), indicating thatin the receptor clusters, the former positions are
more closely located to each other. A very highcrosslinking efficiency at position 118 might
account for the failure of the A118C protein to
support swarming of HCB339 cells. When
combined with the S36C mutation, all of theresulting double Cys mutant proteins yieldedcrosslinked oligomers as has been shown for the
S36C&D142C protein (26), demonstrating that allthese disulfides in or near the loop crosslink
subunits from different dimers. Prolonged
incubation of the double Cys-mutant protein
S36C&D142C with Cu-phenanthroline resulted inaccumulation of the deduced hexamer over theother oligomers (Fig. 2B). In contrast, theS36C&M116C protein did not exhibit such apattern.
On the basis of the efficiencies ofcrosslinking and the assumption of a “trimer ofdimers” unit (24, 38-40), we propose a relative
arrangement of three Tar dimers in a receptor
array (Fig. 3). This model, albeit being only
qualitative without precise distance andorientation, can predict the differential patterns ofcrosslinked oligomers. The S36C&D142C
protein would allow the formation of disulfide
bonds between all six subunits in the model,
whereas the S36C&M116C protein cannot form acrosslinked hexamer: only two Cys residues of thethree “inner” subunits can be crosslinked.
Differential effects of receptor amidation on
crosslinking at various positions.—To examine
the effect of covalent modification on thearrangement of receptor dimers in a cluster, we
introduced mutations into the four methylationsites, Gln295, Glu302, Gln309 and Glu491(collectively referred to as QEQE), of each
Cys-replaced Tar to yield three amidation states:
fully deamidated (EEEE), intermediate (QEQE)
and fully amidated (QQQQ) (Fig. 1). It has been
established that a Gln residue mimics amethylated Glu residue (27, 28) and that receptor
methylation (amidation) increases the mobility ofthe protein in SDS-PAGE. The representative
gel is shown in Fig. 4, demonstrating thatamidation has different effects on crosslinking at

Results
Receptor dimers are organized into a well-definedarray—To probe the relative orientation ofreceptor dimers in a polar cluster, we
systematically introduced Cys residues within ornear loop 2-3 of the periplasmic domain of theaspartate chemoreceptor Tar (Fig. 1). The loop
sticks out from the dimer in the crystal and might
constitute an interdimer contact surface. The
resulting proteins were expressed in HCB339 cells,
which lack all receptors, at levels similar to that ofchromosome-encoded wild-type Tar (data notshown). Cells expressing any mutant Tar except
Tar-A118C, which caused growth retardation,
swarmed as fast as those expressing wild-type Tar(data not shown), indicating that the Cys
substitutions, except A118C, by themselves do notseverely affect receptor function.
To avoid any complexity from covalentmodification, the mutant proteins were thenexpressed in HCB436 cells, which lack all
receptors, the methylesterase CheB, and the
methyltransferase CheR. Immunoblotting
revealed that all of them formed crosslinked
dimers in the presence of Cu-phenanthroline invivo (data not shown), which are deduced to result
from crosslinking between native dimers since thedouble mutants with S36C (located at the subunit
interface within a dimer) yielded crosslinked
oligomers in the presence of Cu-phenanthroline(data not shown). In the absence ofCu-phenanthroline, the crosslinking efficiencies at
positions in helix 3 (A118C-I123C) exhibited aperiodicity that is consistent with thewater-accessibility of an α-helix packed against
the rest of the protein (12, 37) (Fig. 2A, C). The
variation of the mobility of the dimers on the
SDS-PAGE gels should be due to their shape: thecloser the crosslinking point to the midpoint of themolecule, the slower the crosslinked dimer would
migrate in the gel matrix. Along with thedependence of interdimer crosslinking on thepresence of CheA and CheW (data not shown andref. 26), this pattern supports the idea that the invivo interdimer crosslinking of Tar reflects its

3

different positions. It should be noted that boththe concentration of Cu-phenanthroline and theamount of protein were optimized for each Cys
position to detect differences among thedifferentially amidated variants. The assay was
triplicated and the crosslinking efficiencies were
quantified (Fig. 4B). The Cys residues showed
different patterns, which can be divided into threeclasses: the efficiency of crosslinking at a given
position was highest at the (i) QQQQ, (ii) EEEEor (iii) QEQE state, whereas crosslinking at S36C
was not detectably changed. The results are
summarized by color-coded positions in thethree-dimensional structure of Tar (Fig. 5).
The positions showing similar effects ofmethylation (class i, ii or iii) tend to cluster in thethree-dimensional structure, lining up along thelong axis (Fig. 5). Since an efficiency of
disulfide formation at a given position is highly
sensitive to the distance between two thiol groups,
the simplest model to account for the pattern may
be that receptor amidation (methylation) inducesslight rotational movement of Tar dimers either
around the axis of symmetry of the dimer (Fig.
6A) or the "trimer of dimers" (Fig. 6B). Since
receptor methylation does not affect crosslinking
at the cytoplasmic trimer contact (25), it isnecessary to assume a twist at somewhere
between the cytoplasmic trimer contact and thetransmembrane region in the former model.
Such a twist might occur within the structurally
undefined HAMP domain, which connects thesecond transmembrane to the first methylation
helix. The latter model does not require any
twist within a dimer but involves a larger
movement.
Effects of attractant binding oppose those ofamidation—We also examined the effect of
α-methyl-DL-aspartate (MeAsp), a
non-metabolizable analog of aspartate, oninterdimer crosslinking. It has already beenshown that MeAsp does not affect intradimer
crosslinking at S36C but decreases interdimer
crosslinking at D142C without any detectablechange in receptor localization (26), although, ona longer time scale, the addition of an attractant
decreases receptor localization (41). We foundthat MeAsp either increases (S109C and P112C)
or decreases (N122C) interdimer crosslinking
depending on the position of the introduced Cys(Fig. 7). For any mutant tested, the effect of
MeAsp on crosslinking was roughly opposite tothat of receptor methylation. The reciprocal
effects of attractant and methylation are consistent
with previous two-state models of receptor
signaling and adaptation (42-45).
Receptor amidation attenuated attractant effects
on crosslinking—To examine whether the
dimer-to-dimer interaction of Tar is involved ingain control by covalent modification, we
examined the effect of the MeAsp concentration
on interdimer crosslinking. The representative
results are shown in Fig. 8. In this assay,
variations were too high to quantify thecrosslinking efficiencies, but the results werequalitatively reproducible. For N122C, thethreshold concentration of MeAsp for decreasing
crosslinking of the EEEE form was at least103-fold higher than that for the QQQQ form.
Similar results were obtained for D142C (data notshown). By contrast, the EEEE form of the
P112C mutant was crosslinked even in theabsence of MeAsp at an efficiency similar to thatin the presence of saturating concentrations of
MeAsp, presumably because the fully
demethylated state mimics attractant binding atleast to some extent, while crosslinking of the
QQQQ form was increased only in the presence of
1 mM MeAsp. Similar results were obtained for
S109C (data not shown). Thus, the aspartate
effects (regardless of polarity) on interdimer
crosslinking are attenuated by covalentmodification (amidation) of Tar, suggesting thatgain control by covalent modification of thechemoreceptor involves modulation ofarrangement or packing of the receptor array.
Discussion
The structures of the intact chemoreceptors,
the intact kinase CheA, and their complex with theadaptor CheW remain unresolved, which has
impeded understanding of the mechanisms ofsignaling through the chemoreceptor-CheW-CheA
cluster. The systematic disulfide crosslinking
assays presented in this study have led to thefollowing key findings about chemoreceptor
clustering and its physiological significance: (i)chemoreceptor dimers are organized into awell-defined array in E. coli cells; (ii) receptor

4amidation and attractant binding have roughly
opposite effects on the arrangement, packing
and/or dynamics of receptor dimers within thearray; and (iii) increasing levels of amidation
attenuate effects of attractant binding, hence
presumably decreasing signal gain (Fig. 9). In
essence, the receptor array is in an equilibrium
between the kinase ON and OFF states, which arefavored by methylation (amidation) and attractant
binding, respectively. We propose that receptor
methylation controls signal gain by rearranging
receptor dimers (e.g. inducing a small rotational
displacement) with a polarity opposite to
attractant binding and restricting theattractant-induced rearrangement of receptor
dimers.
It has recently been shown that receptor
methylation (amidation) slightly increases its
polar localization (31-33). Although such asmall increase cannot account for adaptation,amidation would enhance interdimer crosslinking
at all positions. The differential effects oncrosslinking at various positions argue thatenhanced localization or clustering is not a major
effect of receptor amidation but that it alters thearrangement or packing of dimers within a

pre-formed cluster. By contrast, crosslinking ofreceptor dimers at the cytoplasmic "trimer
contact" is not affected by receptor amidation oreven by the presence or absence of CheA and
CheW (24, 25). We therefore suspect that the
proposed arrangement (Fig. 3) represents three
dimers from neighboring "trimer of dimers" units
rather than from within a single unit, which isconsistent with previous models that assume awell-defined receptor array (40).
Methylation may also modulate structuraldynamics of the receptor. In fact, the addition of
MeAsp induced little change in crosslinking of the
EEEE forms of the S109C and P112C proteins.
This result is consistent with the suggestion thatthe unmodified chemoreceptor is more dynamic
than the amidated one (40). It remains unclear
how the rearrangement of receptor dimers canregulate the CheA kinase activity. Nevertheless,our results shed new light on the significance ofreceptor clustering or receptor quaternary
structures in signal transduction. Moreover, thecrosslinking technique may be applied to isolate
receptor oligomers or larger clusters for structural
studies.

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FOOTNOTES*We thank Toshiharu Yakushi for helpful discussion and encouragement. This work was supported inpart by grants-in-aid for scientific research to I. K. from the Japan Society for the Promotion of
Science and from the Institute for Advanced Research, Nagoya Univerisy.
The abbreviations used are: Cu-phenanthroline, Cu(II)(o-phenanthroline)3; EDTA,
ethylenediaminetetraacetic acid; 2ME, 2-mercaptoethanol; NEM, N-ethylmaleimide
FIGURE LEGENDS
Fig. 1. Disulfide scanning of the periplasmic domain of the aspartate chemoreceptor Tar. A, The
three-dimensional structure of the periplasmic fragment of Salmonella Tar (12). Residues replaced by
Cys are marked by balls. B, A schematic illustration of the positions of the introduced Cys residues.
Wild-type Tar is devoid of Cys residues. Residues (K108-I123) within or near loop 2-3, a potential
interdimer interface, were systematically replaced by Cys. Substitutions of residue S36 at the subunit
interface within the Tar dimer and residues D142, Y143 and G144 at the external surface of the dimer

6

were described previously (26). Filled circles indicate the four methylation sites (Q295, E302, Q309,
and E491). H, L and TM denote α-helices, loops and transmembrane helices, respectively. Although
the cytoplasmic domain of Tar is larger than the periplasmic domain, it has been simply represented by asingle box in the diagram.
Fig. 2. Detection of in vivo disulfide-crosslinked products of Cys-replaced Tar proteins. A, B, Single
Cys mutant proteins (A) and double Cys mutant proteins (B) were expressed in HCB436 cells anddetected by immunoblotting. Crosslinking of the single Cys mutant proteins was examined in theabsence of Cu-phenanthroline, whereas cells expressing the S36C&D142C or S36C&M116C protein
were incubated for 15 or 20 minutes in the presence of Cu-phenanthroline. C, Quantification of thecrosslinking efficiencies of the single Cys mutant proteins. The crosslinking efficiency (in percentage)of a given Tar mutant protein was defined as a proportion of the crosslinked dimers to the total amount.
Crosslinking assays were at least triplicated with independent transfromants, resulting in consistentpatterns with varied absolute crosslinking efficiencies. Shown here is the representative result.
Fig. 3. Proposed arrangement of three Tar dimers in a receptor array. The green balls denote thepositions of the Cys substitutions. Position 36 is located at the subunit interface within the Tar dimer,whereas the other substitutions were made on the external surface of the dimer. The more deeply
shaded balls indicate positions at which a higher efficiency of crosslinking was observed. Left panel:
top view (i.e. looking down from the outer membrane); right panel: bottom view (i.e. looking up fromthe cytoplasm).
Fig. 4. Differential effects of receptor modification on interdimer crosslinking at different positions.
A, Detection of crosslinked dimers of the three different amidation variants (EEEE: 4E, QEQE: QE,
QQQQ: 4Q) of each Cys-carrying mutant protein. Both the concentration of Cu-phenanthroline and theamount of protein were optimized for each Cys position to detect differences among the differentiallyamidated variants. Note that amidation increases the mobility of the receptor protein in SDS-PAGE.
B, Quantification of the crosslinking efficiencies of mutant Tar proteins of the three different amidation
variants. The crosslinking efficiency (in percentage) of a given Tar mutant protein was defined as aproportion of the crosslinked dimer (on a non-reducing gel) to the total amount (on a reducing gel).
For each mutant, crosslinking assays were triplicated with three independent transfromants and the meanand standard deviation values ware plotted. The P114C and V117C proteins were not included sinceeach of these mutant proteins yielded two separate bands that can be taken as the crosslikned dimer.
Fig. 5. Classification of the Cys positions with respect to the differential effects of receptor amidation
on Cys crosslinking. The Cys residues were placed onto the three-dimensional structure and divided
into three classes: the efficiency of crosslinking at a given position was highest when the amidation stateof the protein was QQQQ (red), EEEE (blue) or QEQE (magenta). Crosslinking at position 36 (yellow)
was not detectably changed by amidation. The P114C and V117C proteins formed two dimer bands,
and the positions of the Cys residues have been marked with pink balls. Left top panel: top view; left
bottom panel: bottom view; right panel: side view.
Fig. 6. Models for modulation of the arrangement of tar dimers by amidation/methylation. Receptor
modification is assumed to induce slight rotational movement around the two-fold symmetry axis of eachdimer (A) or the symmetry axis of each "trimer of dimers" unit (B). The fully deamidated/demethylated(4E) and fully amidated (4Q) states are shown in the left and right panels, respectively. The arrows
indicate the direction of rotation induced by amidation/methylation (left panels) ordeamidation/demethylation (right panels). Note that the displacements are exaggerated for clarity.
Fig. 7.

Differential effects of MeAsp on interdimer crosslinking at various positions. MeAsp (1 mM)

7

increased (S109C, P112C) or decreased (N122C, D142C) the formation of crosslinked Tar dimers in
HCB436 cells. Note that the intermediate (QE) variants of the proteins were used in these assays. The
optimal concentration of Cu-phenanthroline for the detection of the effect of MeAsp was determined foreach position.
Fig. 8. Attenuation of the effects of MeAsp on interdimer crosslinking by receptor amidation.
Crosslinking at P112C and N122C was examined in the presence (200 µM) and absence ofCu-phenanthroline, respectively.
Fig. 9. A model of gain control by covalent modification of the receptors. At a cell pole, thechemoreceptors forms clusters that are made of "trimer of dimers" units (top panel). Our results
demonstrate that these units are organized into a well-defined array (middle panel). Two-state modelsof the chemoreceptor function assume two extreme states: one activating and the other inactivating CheA
(kinase ON and OFF states, respectively). The observed crosslinking is consistent with the notion thatmethylation (amidation) counteracts attractant binding; attractant binding favors the OFF state, whereas
methylation favors the ON state. The results also suggest that receptor methylation restricts therearrangement (rotation) of the dimers by attractant binding (denoted by the longer light blue arrows forthe demethylated state and the shorter arrows for the methylated state), leading to a smaller gain for thesame input signal (bottom panel). Higher levels of methylation may also restrict structural fluctuationsof the Tar molecule (40).
LEGENDS TO SUPPLEMENTAL MATERIALSSUPPLEMENTAL VIDEO 1
Animation depicting proposed methylation-induced rotational
displacements of chemoreceptor dimers around their symmetry axes. The animation begins with the
EEEE state and ends with the QQQQ state. The residues, for which Cys was substituted, are colored as
described in the legend to Fig. 5. Note that the displacements are exaggerated for clarity.
SUPPLEMENTAL VIDEO 2
Animation depicting proposed methylation-induced rotational
displacements of "trimer of dimers" units of the chemoreceptor around their symmetry axes. The
animation begins with the EEEE state and ends with the QQQQ state. The residues, for which Cys was
substituted, are colored as described in the legend to Fig. 5. Note that the displacements are
exaggerated for clarity.

8

A

B

Irieda et al. Fig. 1A

B

0 µM Cu2+
H2

L2-3

H3

2mer

L3-4

200 µM Cu2+(min) 1520
36 36 36 36& & & &
142116 142 116
8mer
6mer
4mer

3mer
2mer(inter)2mer(intra)
1mer
+2ME

1mer

C
60
50
40
30
20
10
0

Cys-replaced residues

Irieda et al. Fig. 2

M116C

D142C

D142C

M116C

M116C
D142C

M116C

Irieda et al. Fig. 3

A[Cu2+]
(µM)

10
S36C
K108C
4E QE 4Q 4E QE 4Q

200
S109C
M110C
4E QE 4Q 4E QE 4Q

0A111C
4E QE 4Q

200
0
P112C
L113C
P114C
E115C
M116C
4E QE 4Q 4E QE 4Q 4E QE 4Q 4E QE 4Q 4E QE 4Q

200
T119C
S120C
4E QE 4Q 4E QE 4Q

0
R121C
N122C
4E QE 4Q 4E QE 4Q

2mer

1mer
+2ME

[Cu2+]
0
V117C
A118C
(µM)

4E QE 4Q 4E QE 4Q

200
10
I123C
D142C
4E QE 4Q 4E QE 4Q

200
Y143C
G144C
4E QE 4Q 4E QE 4Q

2mer

1mer
+2ME

B
demethylated (4E) intermediate (QE) methylated (4Q)

80
70
60
50
40
30
20
10
0

Cys-replaced residues

Irieda et al. Fig. 4

Irieda et al. Fig. 5

A

4E

4Q

B
4E

4Q

Irieda et al. Fig. 6

[Cu2+] (µM)

200
S109C P112C
MeAsp - + - +0200
N122C D142C
+
- +

2mer

1mer
+2ME

Irieda et al. Fig. 7

[MeAsp] (mM)

N122C
4E
QE
4Q
0 10-3 10-1 100 0 10-3 10-1 100 0 10-3 10-1 100

2mer
1mer
+2ME

[MeAsp] (mM)

P112C
4E
QE
4Q
0 10-3 10-1 100 0 10-3 10-1 100 0 10-3 10-1 100

2mer
1mer
+2ME

Irieda et al. Fig. 8

MeAspmethylation

Receptor array
MeAsp
Tar
methylation

MeAsp
MeAsp

demethylated

methylatedInterface of three “trimer of dimers” units

Irieda et al. Fig. 9