Oxidized human neuroglobin acts as a heterotrimeric Galpha protein ...

1

Oxidized human neuroglobin acts as a heterotrimeric

Galpha protein guanine nucleotide dissociation inhibitor

Keisuke Wakasugi*†‡, Tomomi Nakano†, and Isao Morishima*‡

*Department of Molecular Engineering, Graduate School of Engineering, Kyoto University,

Kyoto 606-8501, Japan; †Precursory Research for Embryonic Science and Technology (PRESTO),

Japan Science and Technology Corporation (JST).

‡To whom correspondence should be addressed. e-mail: [email protected]

u.ac.jp

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 14, 2003 as Manuscript M305519200 by guest on M

arch 22, 2018http://w

ww

.jbc.org/D

ownloaded from

2

RUNNING TITLE

Oxidized human Ngb as a GDI

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

ABSTRACT

Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that is expressed in the

brain and can reversibly bind oxygen. It has been reported that Ngb expression levels increase in

response to oxygen deprivation, and that it protects neurons from hypoxia in vitro and in vivo.

However, the mechanism of this neuroprotection remains unclear. In the present study, we tried to

clarify the neuroprotective role of Ngb under oxidative stress in vitro. By surface plasmon

resonance, we found that ferric Ngb, which is generated spontaneously as a result of the rapid

autoxidation, binds exclusively to the GDP-bound form of the α subunit of heterotrimeric G

protein (Gα�). In GDP dissociation assays or GTPγS binding assays, ferric Ngb behaved as a

guanine nucleotide dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP.

The interaction of GDP-bound Gαi with ferric Ngb will liberate Gβγ, leading to protection

against neuronal death. In contrast, ferrous ligand-bound Ngb under normoxia did not have GDI

activities. Taken together, we propose that human Ngb may be a novel oxidative stress-

responsive sensor for signal transduction in the brain.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

INTRODUCTION

Neuroglobin (Ngb) is a recently discovered globin found in the vertebrate brain that has a

high affinity for oxygen (1-3). Globins are iron porphyrin complex (heme)-containing proteins

that bind reversibly to oxygen and as such, play an important role in respiratory function. They

have been found in many taxa including bacteria, fungi, plants, and animals (4). The two major

globins that have been described in vertebrates are hemoglobin and myoglobin. Hemoglobin (Hb),

which consists of four subunits that cooperatively bind oxygen, is present in red blood cells where

it is responsible for transporting oxygen from the lungs to the tissues (5). Myoglobin (Mb) is a

monomeric intracellular globin that stores oxygen in muscle tissue and facilitates its diffusion

from the periphery of the cell to mitochondria, which use it during oxidative phosphorylation (6).

Although Ngb shares only 21 to 25 % sequence identity with vertebrate Hb and Mb, it conserves

the key amino acid residues that are required for Hb and Mb function (1). Like Hb and Mb, Ngb

can reversibly bind oxygen (1,7,8). The iron atom in the heme prosthetic group of each globin

normally exists in either the ferrous (Fe2+) or ferric (Fe3+) state. In the absence of exogenous

ligands, the ferric and ferrous forms of Ngb are hexacoordinated with the endogenous protein

ligands, distal histidine and proximal histidine (7) (Fig. 1). Oxygen (O2) or carbon monoxide

(CO) can displace the distal histidine of ferrous Ngb to produce ferrous oxygen-bound Ngb

(ferrous-O2 Ngb) or ferrous carbon monoxide-bound Ngb (ferrous-CO Ngb) (7). On the other

hand, Hb and Mb are normally hexacoordinated in the ferric state, with a water molecule

coordinated to iron and pentacoordinated in the ferrous form, leaving the sixth position empty and

available for the binding of exogenous ligands such as O2 and CO.

The mammalian brain accounts for up to 20% of the total oxygen consumption even though

it constitutes only 2% of total body weight, and it is the most sensitive organ to the effects of

tissue hypoxia (9). Ngb is widely expressed in the cerebral cortex, hippocampus (CA1, CA2, CA3,

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

and CA4, especially in the pyramidal layer), thalamus, hypothalamus, and cerebellum (1,3,10) of

the rat brain. Recently, it has been suggested that Ngb plays a role in the neuronal response to

hypoxia and ischemia (11,12). Ngb expression was reported to increase in response to neuronal

hypoxia in vitro and focal cerebral ischemia in vivo (11,12). Neuronal survival following hypoxia

was reduced by inhibiting Ngb expression with an antisense oligodeoxynucleotide and was

enhanced by Ngb overexpression, supporting the notion that Ngb protects neurons from hypoxic-

ischemic insults (11). Moreover, Ngb protected the brain from experimental stroke in vivo (12).

A possible mechanism by which Ngb protects these neurons is by functioning as an O2

carrier, facilitating the diffusion of O2 to the mitochondria within cells that are engaging in active

aerobic metabolism, in a manner similar to the way Mb acts in muscle cells. However, Ngb has

been estimated to comprise less than 0.01% of the total protein content in the brain (1). The low

concentration (in the micromolar range) of Ngb in brain tissue perhaps argues against its role in

storing and carrying significant amounts of O2. On the other hand, local concentrations of Ngb

may reach sufficiently high levels to allow it to regulate local oxygen consumption (10,13).

Finally, though debatable, the affinity of Ngb for oxygen may be so high as to prevent its release

under physiological conditions (7,8). Thus, the mechanism by which Ngb affords neuroprotection

under oxidative stress conditions such as ischemia and reperfusion remains unclear.

The objective of this study was to investigate whether Ngb has novel functions that are

related to neuroprotective roles under oxidative stress. Online BLAST searches were performed

via the website of the National Center for Biotechnology Information (NCBI) (Conserved

Domain

Database,http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi;http://www.ncbi.nlm.nih.gov/BL

AST). These analyses revealed that human Ngb has 25-35 % amino acid sequence homology with

regulators of G protein signaling (RGS) and RGS domains of G protein-coupled receptor kinases

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

(GRK) (Fig. 1). The protein that most closely resembles Ngb (24 % amino acid identity) is GRK4

(Fig. 1). RGS and GRK proteins are modulators of heterotrimeric G proteins (14-17).

Heterotrimeric G proteins (G proteins) consist of an α subunit (Gα) with GTPase activity and a

βγ dimer (Gβγ), and belong to a family of proteins, whose signal transduction function depends

on the binding of guanine nucleotides (18-23). Ligand- or signal-activated G protein-coupled

receptors (GPCRs) induce GDP release from a Gα subunit, which is followed by the binding of

GTP. Binding of GTP to Gα “turns on” the system and causes conformational changes that result

in dissociation of the GTP-bound Gα from both the receptor and Gβγ. The GTP-bound Gα and

Gβγ can then regulate the activity of different effector molecules, such as adenylyl cyclase,

phospholipase Cβ, and ion channels. Signal transduction is “turned off” by the intrinsic GTPase

activity of the Gα protein, that hydrolyzes the bound GTP to GDP, inducing the reassociation of

GDP-bound Gα with Gβγ. The on/off G protein ratio can be regulated by three groups of protein

modulators: guanine nucleotide exchange factors (GEFs) which stimulate GDP dissociation and

subsequent GTP-binding; guanine nucleotide dissociation inhibitors (GDIs) which inhibit GDP

dissociation; and GTPase-activating proteins (GAPs) which enhance GTP hydrolysis (18-23).

GPCRs play a role as functional analogues of GEFs (18,20,21). GRKs phosphorylate agonist-

activated forms of GPCRs to induce homologous desentsitization of signaling pathways (14,15).

RGS proteins act as GAPs for Gαi or Gαo and play a role in desensitization (16,17).

In the present study, we examined the possibility of interaction of Ngb with Gα by surface

plasmon resonance (SPR) measurements. Ferric Ngb interacted exclusively with Gαi in their

GDP-bound forms. In GDP dissociation assays or GTPγS binding assays, ferric Ngb exhibited

GDI activity, inhibiting the rate of exchange of GDP for GTP by Gαi. Since ferrous ligand-bound

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

Ngb under normoxia did not have GDI activities, human Ngb may function as a novel oxidative

stress-responsive sensor for signal transduction in the brain.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

EXPERIMENTAL PROCEDURES

Samples

Rat myristoylated Gα-subunits (Gαi1, Gαi2, Gαi3, and Gαo; Calbiochem, San Diego, CA) and

bovine Gβγ (Calbiochem) were used. [35S]GTPγS (>1000 Ci/mmol), [8-3H]GDP (10-15

Ci/mmol) and [α-32P]GTP (~3000 Ci/mmol) were purchased from Amersham Pharmacia Biotech

(Buckinghamshire, England).

Preparation of proteins

Amplification of human Ngb cDNA was performed by PCR using human universal Quick-clone

cDNA (Clontech, Palo Alto, CA). Human Ngb cDNA was cloned into plasmid PET20b

(Novagen, Madison, WI) and was sequenced using an ABI 3100 Genetic Analyzer (Applied

Biosystems, Foster City, CA). Overexpression of human Ngb was induced in E. coli strain BL 21

(DE 3) (Novagen) by treatment with isopropyl β-D-thiogalactopyranoside for 4 h. Purification of

Ngb without 6xHis-tag was carried out as follows (24): Soluble cell extract was loaded onto a

DEAE sepharose anion-exchange column equilibrated with 20 mM Tris-HCl pH 8.0. Ngb was

eluted from the column with buffer containing 75 mM NaCl and was further purified by passage

through a Sephacryl S-200 HR gel filtration column. Human Ngb mutants, including a COOH-

terminal tag of six histidine residues (6xHis-tag), were purified on nickel affinity columns

(His•Bind® resin; Novagen) from the supernatant of lysed cells using the protocol provided by

Novagen.

Mass spectrometric measurements of purified Ngbs were performed using MALDI-TOF

mass spectrometry (PerSpective Biosystems VoyagerTM DE PRO-SD, Applied Biosystems), and

Edman degradation was carried out on purified Ngbs using a G1005A protein sequencing system

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

(Hewlett Packard, Palo Alto, CA) at Takara Biomedicals, Inc. in order to determine their NH2-

terminal sequences.

Ferric Ngbs were incubated with 2 mM dithiothreitol (DTT) for 2 h, and then DTT was

removed by chromatography using a PD-10 column (Amersham Pharmacia Biotech). Ferrous-CO

Ngbs were generated after addition of sodium dithionite and CO gas to the DTT-treated ferric

Ngb followed by gel filtration,.

Site-directed mutagenesis

A QuikChangeTM site-directed mutagenesis system (Stratagene, La Jolla, CA) was used to alter

cysteine residues (amino acid residues 46, 55, and/or 120) in human Ngb. The point mutations

were confirmed by DNA sequencing using BigDye Terminator Cycle Sequencing FS (Applied

Biosystems) and an ABI 3100 Genetic Analyzer (Applied Biosystems).

Surface plasmon resonance (SPR) experiments

SPR measurements were performed on a BIAcore® X Instrument (Biacore, Uppsala, Sweden).

Rat myristoylated Gα-subunit (Gαi, Gαi2, Gαi3, or Gαo) was immobilized on the surface of a

CM5 sensor chip using an amine coupling kit (Biacore) according to the instructions of the

manufacturer. Activation of the carboxymethylated dextran in the CM5 sensor chip was carried

out by mixing equal volumes of 400 mM N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide in

water and 100 mM hydrochloride/N-hydroxysuccinimide in water, and injecting the mixture into

the instrument at 10 µl/min for 7 min. This was followed by the injection of 5 µg/�l of Gα

protein dissolved in 10 mM acetate buffer (pH 4.5) over the activated surface of the sensor chip

for 7 min at a flow-rate of 10 µl/min. The unreacted sites of the sensor chip were masked by the

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

injection for 7 min of 1 M ethanolamine, pH 8.5. After immobilization, non-specifically bound

protein was removed by washing with running buffer (10 mM Hepes, 150 mM NaCl, 0.005 %

Tween20, pH 7.4) until the value of the resonance units (RU) became nearly constant.

All binding experiments were performed at 25 °C at a flow rate of 5 µl/min. Ferric or ferrous-

CO Ngb in the running buffer was injected for 5 min, during which association occurred.

Dissociation then took place in the running buffer over the next 10 min. The BIAcore response

was expressed in relative RU i.e., the difference in response between flow cell with immobilized

protein and the control flow channel. 1000 RU corresponded to 1 ng/mm2 of bound ligand. For

binding analyses in the presence of guanine nucleotides, the running buffer containing 5 mM

MgSO4 and either 500 µM GDP, or 500 µM GDP plus 500 µM AlCl3 and 10 mM NaF was

loaded for 60 min to allow binding of guanine nucleotide to immobilized Gα, after which the

samples were injected. After each binding cycle, the sensor chip was regenerated with 5 µl of

0.05 % SDS in the running buffer and was washed with running buffer for 5-10 min prior to the

next injection. Experimental curves (sensorgrams) were analyzed by means of the BIAevaluation

3.1 software package using the model A + B ⇔ AB to estimate the association and dissociation

rate constants ka and kd.

GTPγS binding assays

100 nM Gαi1 or Gαo was incubated for 3 min at 25 °C in buffer A (20 mM Tris-HCl, 100 mM

NaCl, and 10 mM MgSO4 at pH 8.0) with 10 µM GDP in the absence or presence of Ngb (5 µM).

Binding assays were initiated with additions of 50 nM [35S]GTPγS (>1000 Ci/mmol). Aliquots

(10 µl) were withdrawn from the binding mixtures and were passed through nitrocellulose filters

(0.45 µm) (Millipore, Bedford, MA). The filters were then washed three times with 1 ml of ice-

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

cold buffer A and were counted in a liquid scintillation counter (LSC-6100; Aloka, Tokyo, Japan).

The apparent rate constant (kapp) values for the binding reactions were calculated by fitting the

data to the following equation: GTPγS binding (%) = 100 % x (1-e-kt).

GDP dissociation assays

Gαi1 complexed with [3H]GDP (0.3 µM) was prepared by incubating 0.3 µM Gαi1 with 2 µM

[3H]GDP in buffer A for 1.5 h at 25 °C. Excess unlabeled GTP or GDP (200 µM) was added to

monitor dissociation of [3H]GDP from Gαi1 in the absence or presence of Ngb (5 µM). Aliquots

were withdrawn at the indicated times and were passed through nitrocellulose filters (0.45 µm)

(Millipore, Bedford, MA). The filters were then washed three times with 1 ml of ice-cold buffer

A and were counted in a liquid scintillation counter (LSC-6100; Aloka). As for preparation of

Gαi1 complexed with [α-32P]GDP (1 µM), 1 µM Gαi1 and 2 µM [α-32P]GTP were incubated in

buffer A for 1.5 h at 25 °C. Experiments using Gαi1 complexed with [α-32P]GDP were also

performed, as described above.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

RESULTS

SPR detection of human ferric Ngb binding to Gα

Proteins that interacted with human Ngb were sought by SPR experimentation. SPR is a

powerful tool for real time measurement of direct protein-protein interactions that do not require

labeling of the proteins. We covalently coupled rat Gαi, which is highly expressed in the brain

(18), to a sensor chip. As positive and negative controls we used Gβγ and Mb, respectively, and

confirmed that Gβγ interacts with GDP-bound Gαi but Mb does not bind to Gαi by SPR

(Supplement). Then we characterized the interaction between Ngb and Gαi by SPR. A

representative sensorgram in Fig. 2A shows that the resonance response reflecting Gαi-ferric Ngb

interaction occurred in an analyte concentration-dependent manner. In the association phase (0 ~

300 s), the intensity of SPR increased, indicating that ferric Ngb bound to Gαi specifically, while

in the dissociation phase (300 ~ 900 s), the intensity of SPR decreased, indicating that ferric Ngb

dissociated from the immobilized Gαi. Binding parameters for the interaction of ferric Ngb with

Gαi were determined to be as follows: association rate constant, ka = 5.0 x 102 M-1s-1;

dissociation rate constant, kd = 3.0 x 10-4 s-1; and equilibrium dissociation constant, Kd = kd/ka =

6.0 x 102 nM. No significant resonance signals were obtained from sensor chip surfaces that did

not have attached ligands (data not shown), indicating an absence of nonspecific interactions

between the sensor chip surfaces and analytes.

Next we investigated the possibility of interaction of ferrous-O2 Ngb with Gαi. Since ferrous-

O2 Ngb is unstable and is converted into ferric Ngb very rapidly due to its autoxidation (7), stable

ferrous-CO Ngb was used for SPR experiments. As shown in Fig. 2B, the binding affinity of

ferrous-CO Ngb to Gαi was significantly low (Kd > 1mM) as compared with that of ferric Ngb.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

Moreover, further SPR measurements clarified that human ferric Ngb binds to Gαi2, Gαi3,

and Gαo (Kd = 5.8 x 102, 5.5 x 102 and 6.1 x 102 nM, respectively), whereas ferrous-CO Ngb

does not bind them (Kd > 1 mM).

Ferric Ngb interacts exclusively with the GDP-bound form of Gα

Next we investigated guanine nucleotide dependence of the binding of Gαi1 to Ngb by SPR

measurements. As shown in Fig. 2C, ferric Ngb bound to Gαi even in the presence of Mg2+ and

GDP. The binding parameters (ka = 1.1 x 103 M-1s-1, kd = 6.8 x 10-4 s-1, Kd = 6.0 x 102 nM) were

almost the same as those seen in the absence of Mg2+ and GDP. Aluminium tetrafluoride (AlF4-),

together with Mg2+, can interact with Gαi1-bound GDP and mimic GTP, and thereby activate

Gαi1 (21,22). In the presence of Mg2+, GDP and AlF4-, ferric Ngb did not bind to the activated

Gαi (Fig. 2C). Therefore, human ferric Ngb clearly interacts exclusively with the inactive (GDP-

bound) form of Gαi1.

Effects of Ngb on GTPγS binding to Gα

Since our SPR data suggested that human ferric Ngb interacts with GDP-bound Gαi1 but not

interact with activated GTP-bound Gαi1, we hypothesized that Ngb may function as a GEF or

GDI for Gαi1. To determine whether ferric Ngb functions as a GEF or a GDI, we performed

GTPγS (a nonhydrolyzable analog of GTP) binding experiments. Increased GTPγS binding to

Gαi1 would imply ferric Ngb is a GEF, whereas decreased binding would imply that ferric Ngb is

a GDI.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

As shown in Fig. 3A, Gαi1 bound GTPγS due to spontaneous guanine nucleotide exchange

(kapp = 0.081 min-1). In the presence of ferric Ngb, the rate of GTPγS binding to Gαi1 was reduced

6.2-fold (kapp= 0.013 min-1) (Fig. 3A), implying that ferric Ngb functions as a GDI for Gαi. On

the other hand, ferrous-CO Ngb had no effect on the GTPγS binding (kapp = 0.078 min-1) (Fig.

3A).

Moreover, ferric Ngb inhibited the rate of GTPγS binding to Gαo by 10-fold, in contrast

ferrous-CO Ngb had no effect (kapp = 0.040, 0.004, and 0.038 min-1 in the absence and presence

of ferric and ferrous-CO Ngb, respectively) (Fig. 3B). These results imply that ferric Ngb is a

GDI for Gαo as well as Gαi.

Ferric Ngb acts as a GDI

We then addressed the mechanism by which human ferric Ngb inhibited GTPγS binding to

Gαi and Gαo. The inhibition of GTPγS binding to Gαi/o by ferric Ngb may reflect a reduction in

the rate of nucleotide exchange. To examine the effects of ferric Ngb on the release of GDP from

Gαi1, we measured the rates of GDP dissociation in the absence or presence of ferric Ngb. In the

presence of an excess amount of unlabeled GTP, [3H]GDP release from [3H]GDP-bound Gαi1

was inhibited by ferric Ngb (Fig. 4A). The inhibition of GDP dissociation by ferric Ngb suggests

that ferric Ngb diminished the rates of spontaneous GTPγS binding to Gαi and Gαo by blocking

the GDP release. In other words, ferric Ngb functions as a GDI for Gαi.

The most representative GDIs for heterotrimeric G proteins share conserved sequence repeats

named the G protein regulatory (GPR) (25) or GoLoco motifs (26). One of the family, Purkinje

cell protein-2 (Pcp2), can modulate GDP binding to Gαo and Gαi (27,28). In the presence of

excess unlabeled GTP, Pcp2 preferentially interacts with the GDP-bound conformation of Gα and

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

serves exclusively as a GDI as does human ferric Ngb (28,29). On the other hand, in the presence

of excess unlabeled GDP, Pcp2 was reported to stimulate GDP release from Gαo (27). To further

characterize properties of Ngb as a GDI, we performed GDP dissociation assays in the presence

of excess GDP. As shown in Fig. 4B, ferric Ngb stimulated [3H]GDP release from [3H]GDP-

bound Gαi1 in the presence of an excess amount of unlabeled GDP, suggesting that the

mechanism of ferric Ngb as a GDI is similar to that of Pcp2. Experiments using [α32P]-GDP

instead of [3H]-GDP also supported these results (data not shown).

Functional analyses of Ngb with an intra- or intermolecular disulfide bond

Cysteine (Cys) residues are particularly sensitive to oxidation by almost all forms of reactive

oxygen species during ischemia and reperfusion (30). Under even mild oxidative conditions, Cys

residues are converted to disulfides (31). Human Ngb has three Cys residues at positions 46, 55,

and 120 (Fig. 1). Cys55 and Cys120 are conserved among mammalian Ngbs, whereas Cys46 is

specific for human Ngb. We investigated whether human wild-type Ngb before DTT treatment

forms a disulfide bond. Fig. 5A shows the SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

that was run under nonreducing conditions. The protein treated with DTT migrated slower than

the untreated protein. Since it has been reported that a protein containing an intramolecular

disulfide linkage has a smaller radius of gyration and migrates further down a gel (32), these data

suggest that ferric Ngb forms a disulfide bond between Cys46, Cys55, or Cys120 spontaneously

upon exposure to air.

To investigate the role of a disulfide bond in the functioning of Ngb, Ngb Cys→Ser mutants

[three single mutants (C46S; C55S; C120S), three double mutants (C46S,C55S; C46S,C120S;

C55S,C120S) and a triple mutant (C46S,C55S,C120S)] with C-terminal 6 x His tag were

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

prepared. The C120S Ngb mutant formed a disulfide bond as did wild-type Ngb, while the C46S

and C55S mutants did not (data not shown), suggesting that the intramolecular disulfide bond

between Cys46 and Cys55 is present in human wild-type Ngb. As shown in Fig. 5B, we found that

a double mutant (C55S,C120S) forms a dimer. This dimer was converted into a monomer by

incubation with DTT (Fig. 5B), indicating that the intermolecular disulfide bond Cys46-Cys46 was

present in this mutant.

Next we investigated GDI activities of these Ngbs with the intra- or intermolecular disulfide

bond. As shown in Fig. 6A, human wild-type ferric Ngb with the intramolecular disulfide bond

inhibited GDP release as did the DTT-reduced ferric Ngb. Double mutant (C55S,C120S)

homodimer linked by the intermolecular disulfide bond did not inhibit GDP/GTP exchange of

Gαi1, whereas its DTT-reduced monomeric form inhibited GDP/GTP exchange of Gαi1 (Fig. 6A),

implying that formation of the intermolecular Cys46-Cys46 disulfide bond in the double mutant

blocks binding sites with Gαi1. As a control, triple mutant (C46S,C55S,C120S), which can not

form a disulfide bond, acted as a GDI with or without DTT (Fig. 6A). As shown in Fig. 6B, in the

presence of excess unlabeled GDP, only double mutant (C55S,C120S) homodimer could not

stimulate [3H]GDP release from [3H]GDP-bound Gαi1. These data also imply that residues

around Cys46 are important for GDI activities of human ferric Ngb.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

DISCUSSION

During ischemia and reperfusion, overproduction of nitric oxide (NO) occurs due to

induction of expression of NO synthetases (33). Reactive oxygen species, which have been

identified as central mediators in certain signaling events, are also generated (34). Reaction of

ferrous-O2 Mb or Hb with NO and/or reactive oxygen species converts these proteins to their

ferric forms (35-38). Thus, ferrous-O2 Ngb may become ferric Ngb during ischemia and

reperfusion. In fact, Ngb has been reported to have a surprisingly high rate of auto-oxidation (7).

Moreover, a recent histochemical study suggested that Ngb transcript exists in the brain areas

important for adaptive responses to stressful events and that Ngb and NO synthetase are co-

expressed in a number of nuclei (39). Our results showed that ferric Ngb functions as a novel GDI

for Gαi/o in vitro. The interaction of Gαi with ferric Ngb will liberate Gβγ, leading to protection

against neuronal death. Since ferrous ligand-bound Ngb, which is a normal form under normoxia,

does not have GDI activities, Ngb may act as an oxidative stress-responsive sensor for signal

transduction in the brain (Fig. 7).

Structure and function of Ngb - the hypoxia-regulated sensor

In this study, we showed that human Ngb, which contains weak homology to RGS domains,

functions as a novel GDI in vitro. Human ferric Ngb binds exclusively to the GDP-bound form of

Gαi and inhibits GDP/GTP exchange of Gαi. On the other hand, ferrous-CO Ngb did not interact

with Gαi and did not have GDI activities. Therefore, it is theorized that the regulation of

interaction of Ngb with Gαi is dependent on oxidation/reduction state and ligand binding of Ngb.

Nonsymbiotic plant hemoglobins (nsHbs) have some characteristics that are similar to Ngb

including the fact they are both members of a newly discovered class of "hexacoordinated"

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

globins and are expressed at low levels (1,40-43). Hypoxia induces the expression of nsHbs as

well as Ngb (44,45). While the three-dimensional structure of Ngb has not as yet been determined,

that of a nsHb (rice Hb1) has been determined (40). In the hexacoordinated structure (Fe3+) of rice

Hb1, electron density of the Phe B10 in the distal pocket is very low because of substantial

disorder, and it has been suggested that the entire CD corner is disordered because of distal His

binding to the heme iron (40). Crystallographic modeling suggests that ligand binding occurs by

an upward and outward movement of the E helix, a concomitant dissociation of the distal

histidine, a possible repacking of the CD corner and folding of the D helix (40).

It is tempting to speculate that Ngb undergoes large tertiary structural changes around the CD

corner and D helix when its ferrous-O2 form is converted into bis-His conformation

(hemichromogen or hemochromogen), as described for rice Hb1 (40). Furthermore, it can be

speculated that the structural change around the CD corner and D helix triggers changes in

affinity for the binding of Gαi if that binding occurs in the CD region and D helix. In fact, in this

study we have demonstrated that Ngb double mutant (C55S,C120S) homodimer linked by the

intermolecular disulfide bond at Cys46-Cys46 can not function as a GDI, whereas its DTT-reduced

monomeric form acts as a GDI, suggesting that the CD corner in which Cys46 exists is important

for protein-protein interaction between ferric Ngb and Gαi1 and is responsible for GDI activities

for Gα.

It should also be noted that a disulfide bond between Cys46 and Cys55 of Ngb is located at the

CD corner and D helix. Therefore, the S-S bond formed during hypoxia may contribute to the

stabilization of structures near the CD corner and D helix of oxidized Ngb, which is the active

form for signal transduction in the brain, since it has been reported that introduction of a disulfide

bond enhances the thermal and conformational stability of proteins (46,47). Because Cys46 is

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

present in human Ngb but is not present in other mammalian Ngbs, human Ngb might have

evolved to stabilize the active form during hypoxia.

Novel brain-specific signaling pathway under oxidative stress

It has recently been reported that Gαi and Gαo are direct target proteins of reactive oxygen

species generated during ischemia and reperfusion and that they are activated in the absence of

GPCR-mediated signaling (48,49). Reactive oxygen species modify two cysteine residues of Gαi

and Gαo (49). Modification of Gαi and Gαo accelerates GDP release from Gα and increases the

formation of the GTP-bound form of Gα without receptor activation (48,49).

We have shown here that human ferric Ngb, which may be produced under oxidative stress

conditions such as ischemia and reperfusion, functions as a GDI that keeps Gαi or Gαo in its

inactive state as does Pcp-2 (GPR, GoLoco). The interaction of Gαi/o with ferric Ngb under

oxidative stress is a novel brain-specific signaling pathway (Fig. 7). Since GPR/Gαi/o interaction

prevents Gβγ from returning to Gα and thus leads to enhanced Gβγ-dependent signaling (28,50),

human ferric Ngb would selectively shut off signaling pathways linked to Gα effectors and favor

Gβγ effector pathways. The intracellular signal transduction induced by Gβγ protects the cells

against oxidative stress (51): Gβγ stimulate proliferation via mitogen activated protein kinase

(MAPK) pathways and promote cell survival by the activation of phosphotidylinositol-3-kinase

(PI3K). Taken together, the characteristic of human ferric Ngb as a GDI may play important roles

in neuroprotective function of human Ngb in the brain. Further study will be necessary to

understand the physiological significance of the interaction of Ngb with Gα in the brain.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

20

REFERENCES 1. Burmester, T., Weich, B., Reinhardt, S., and Hankeln, T. (2000) Nature 407, 520-523

2. Awenius, C., Hankeln, T., and Burmester, T. (2001) Biochem. Biophys. Res. Commun. 287,

418-421

3. Zhang, C., Wang, C., Deng, M., Li, L., Wang, H., Fan, M., Xu, W., Meng, F., Qian, L., and He,

F. (2002) Biochem. Biophys. Res. Commun. 290, 1411-1419

4. Hardison, R. C. (1996) Proc. Natl. Acad. Sci. USA 93, 5675-5679

5. Dickerson, R. E., and Geis, I. (1983) in Hemoglobin: Structure, function, evolution and

pathology (Benjamin/Cummings, Menlo Park, CA)

6. Wittenberg, B. A., and Wittenberg, J. B. (1989) Annu. Rev. Physiol. 51, 857-878

7. Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V., Aerts, T., Marden, M.

C., Caubergs, R., and Moens, L. (2001) J. Biol. Chem. 276, 38949-38955

8. Trent, J. T. 3rd, Watts, R. A., and Hargrove, M. S. (2001) J. Biol. Chem. 276, 30106-30110

9. Ereciñska, M., and Silver, I. A. (2001) Respir. Physiol. 128, 263-276

10. Reuss, S., Saaler-Reinhardt, S., Weich, B., Wystub, S., Reuss, M. H., Burmester, T., and

Hankeln, T. (2002) Neuroscience 115, 645-656

11. Sun, Y., Jin, K., Mao, X. O., Zhu, Y., and Greenberg, D. A. (2001) Proc. Natl. Acad. Sci.

USA 98, 15306-15311

12. Sun, Y., Jin, K., Peel, A., Mao, X. O., Xie, L., and Greenberg, D. A. (2003) Proc. Natl. Acad.

Sci. USA 100, 3497-3500

13. Schmidt, M., Gieβl, A., Laufs, T., Hankeln, T., Wolfrum, U., and Burmester, T. (2003) J.

Biol. Chem. 278, 1932-1935

14. Pitcher, J. A., Freedman, N. J., and Lefkowitz, R. J. (1998) Annu. Rev. Biochem. 67, 653-692

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

21

15. Penn, R. B., Pronin, A. N., and Benovic, J. L. (2000) Trends Cardiovasc. Med. 10, 81-89

16. Zheng, B., De Vries, L., and Farquhar, M. G. (1999) Trends Biochem. Sci. 24, 411-414

17. Wieland, T., and Chen, C.-K. (1999) Naunyn-Schmiedeberg’s Arch. Pharmacol. 360, 14-26

18. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649

19. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127

20. Simon, M. I., Strathmann, M. P., and Gautam, N. (1991) Science 252, 802-808

21. Hepler, J. R., and Gilman, A. G. (1992) Trends Biochem. Sci. 17, 383-387

22. Sprang, S. R. (1997) Annu. Rev. Biochem. 66, 639-678

23. Vetter, I. R., and Wittinghofer, A. (2001) Science 294, 1299-1304

24. Kriegl, J. M., Bhattacharyya, A. J., Nienhaus, K., Deng, P., Minkow, O., and Nienhaus, G. U.

(2002) Proc. Natl. Acad. Sci. USA 99, 7992-7997

25. Takesono, A., Cismowski, M. J., Ribas, C., Bernard, M., Chung, P., Hazard III, S., Duzic, E.,

and Lanier, S. M. (1999) J. Biol. Chem. 274, 33202-33205

26. Siderovski, D. P., Diversé-Pierluissi, M. A., and De Vries, L. (1999) Trends Biochem. Sci. 24,

340-341

27. Luo, Y., and Denker, B. M. (1999) J. Biol. Chem. 274, 10685-10688

28. Natochin, M., Gasimov, K. G., and Artemyev, N. O. (2001) Biochemistry 40, 5322-5328

29. Natochin, M., Gasimov, K. G., and Artemyev, N. O. (2002) Biochemistry 41, 258-265

30. Eaton, P., Byers, H. L., Leeds, N., Ward, M. A., and Shattock, M. J. (2002) J. Biol. Chem.

277, 9806-9811

31. Berlett, B. S., and Stadtman, E. R. (1997) J. Biol. Chem. 272, 20313-20316

32. Uchida, T., Unno, M., Ishimori, K., and Morishima, I. (1997) Biochemistry 36, 324-332

33. Esplugues, J. V. (2002) Br. J. Pharmacol. 135, 1079-1095

34. Dröge, W. (2002) Physiol. Rev. 82, 47-95

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

22

35. Brunori, M. (2001) Trends Biochem. Sci. 26, 209-210

36. Flögel, U., Merx, M. W., Gödecke, A., Decking, U. K. M., and Schrader, J. (2001) Proc. Natl.

Acad. Sci. USA 98, 735-740

37. Joshi, M. S., Ferguson, T. B., Jr., Han, T. H., Hyduke, D. R., Liao, J. C., Rassaf, T., Bryan, N.,

Feelisch, M., and Lancaster, J. R., Jr. (2002) Proc. Natl. Acad. Sci. USA 99, 10341-10346

38. Ouellet, H., Ouellet, Y., Richard, C., Labarre, M., Wittenberg, B., Wittenberg, J., and Guertin,

M. (2002) Proc. Natl. Acad. Sci. USA 99, 5902-5907

39. Mammen, P. P. A., Shelton, J. M., Goetsch, S. C., Williams, S. C., Richardson, J. A., Garry,

M. G., and Garry, D. J. (2002) J. Histochem. Cytochem. 50, 1591-1598

40. Hargrove, M. S., Brucker, E. A., Stec, B., Sarath, G., Arredondo-Peter, R., Klucas, R. V.,

Olson, J. S., and Phillips Jr., G. N. (2000) Structure 8, 1005-1014

41. Kawada, N., Kristensen, D. B., Asahina, K., Nakatani, K., Minamiyama, Y., Seki, S., and

Yoshizato, K. (2001) J. Biol. Chem. 276, 25318-25323

42. Trent, J. T. 3rd, Hvitved, A. N., and Hargrove, M. S. (2001) Biochemistry 40, 6155-6163

43. Burmester, T., Ebner, B., Weich, B., and Hankeln, T. (2002) Mol. Biol. Evol. 19, 416-421

44. Trevaskis, B., Watts, R. A., Andersson, C. R., Llewellyn, D. J., Hargrove, M. S., Olson, J. S.,

Dennis, E. S., and Peacock, W. J. (1997) Proc. Natl. Acad. Sci. USA 94, 12230-12234

45. Sowa, A. W., Duff, S. M. G., Guy, P. A., and Hill, R. D. (1998) Proc. Natl. Acad. Sci. USA

95, 10317-10321

46. Perry, L. J., and Wetzel, R. (1984) Science 226, 555-557

47. Kanaya, S., Katsuda, C., Kimura, S., Nakai, T., Kitakuni, E., Nakamura, H., Katayanagi, K.,

Morikawa, K., and Ikehara, M. (1991) J. Biol. Chem. 266, 6038-6044

48. Nishida, M., Maruyama, Y., Tanaka, R., Kontani, K., Nagao, T., and Kurose, H. (2000)

Nature 408, 492-495

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

23

49. Nishida, M., Schey, K. L., Takagahara, S., Kontani, K., Katada, T., Urano, Y., Nagano, T.,

Nagao, T., and Kurose, H. (2002) J. Biol. Chem. 277, 9036-9042

50. Bernard, M. L., Peterson, Y. K., Chung, P., Jourdan, J., and Lanier, S. M. (2001) J. Biol.

Chem. 276, 1585-1593

51. Schwindinger, W. F., and Robishaw, J. D. (2001) Oncogene 20, 1653-1660

52. Hubbard, S. R., Hendrickson, W. A., Lambright, D. G., and Boxer, S. G. (1990) J. Mol. Biol.

213, 215-218

53. de Alba, E., De Vries, L., Farquhar, M. G., and Tjandra, N. (1999) J. Mol. Biol. 291, 927-939

54. Rost, B., and Sander, C. (1993) J. Mol. Biol. 232, 584-599

55. Tesmer, J. J. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261

56. Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., and Sigler, P. B. (2001)

Nature 409, 1071-1077

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

24

FOOTNOTES

This work was supported in part by Grants-in-Aid 13780532 and 15770085 for Young Scientists

(B) (to K. W.), a Grant-in-Aid 12215077 for Scientific Research on Priority Areas (to K. W.) and

a Grant-in-Aid 12002008 for Specially Promoted Research (to I. M.) from the Ministry of

Education, Culture, Sports, Science and Technology of Japan.

‡To whom correspondence should be addressed. e-mail: [email protected]

u.ac.jp

1The abbreviations used are: Ngb, neuroglobin; ferrous-02 Ngb, ferrous oxygen-bound Ngb;

ferrous-CO Ngb, ferrous carbon monoxide-bound Ngb; G protein, guanine nucleotide-binding

protein; GRK, G protein-coupled receptor kinase; RGS, regulator of G protein signaling; GPCR,

G protein-coupled receptor; GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide

dissociation inhibitor; GAP, GTPase activating protein; DTT, dithiothreitol; SDS-PAGE, sodium

dodecyl sulfate-polyacrylamide gel electrophoresis; SPR, surface plasmon resonance; RU,

resonance units; GTPγS, guanosine 5’-O-(3-thio)triphosphate; Pcp2, Purkinje cell protein-2; GPR,

G protein regulatory; GoLoco, Gαi/o-Loco interaction; GAIP, Gα interacting protein.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

25

FIGURE LEGENDS

FIG. 1. Sequence alignment of human Ngb to RGS domains.

The multiple sequence alignment was performed by Clustal W and manual adjustments. GRK2,

GRK4, GRK5, GRK6, RGS1, RGS4, RGS9 and GAIP share RGS domains. Human myoglobin

(Mb) was used as a representative protein among globin family members. Consensus amino acids

between Ngb and RGS domain are indicated as red letters. Residues highlighted in yellow are

conserved residues that form the hydrophobic core of the RGS domain. Numbers on the left and

right of the sequences correspond to those at the beginning and end of the sequences, respectively.

Gaps in the sequences are indicated by dashes. Boundaries of α helices in human Mb or GAIP,

based on its crystal structure (52,53), are depicted as boxes above or below the sequence,

respectively. The secondary structure prediction for human Ngb obtained with the program PHD

(54) is shown above the sequence (boxes for α-helices and continuous lines for the rest).

Residues in RGS4 and RGS9 that are involved in contacts (< 4.0 �) with Gαi1 and the Gαi/t

chimera, respectively, are blue (55,56). Cysteine residues of Ngb are highlighted in green, and

distal and proximal histidine residues of Ngb are highlighted in purple. The primary sequences

used in the alignment are human myoglobin (Mb, 154 amino acid (aa) protein, accession number

NP_005359), human neuroglobin (Ngb, 151 aa, NP_067080), human GRK2 (689 aa P25098),

human GRK4 (578 aa, P32298), human GRK5 (590 aa, P34947), human GRK6 (576 aa, P43250),

human RGS1 (196 aa, Q08116), human RGS4 (205 aa, P49798), human RGS9 (443 aa,

NP_003826) and human GAIP (217 aa, CAA62919).

FIG. 2. SPR analyses of human Ngb binding to G protein α-subunit Gαi1. A, Concentration

dependence of ferric Ngb on binding affinities with Gαi1. Gαi1 was immobilized to a CM5 sensor

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

26

chip. The immobilization level of Gαi1 was 10000 resonance units (RU). The on and off

processes for ligand binding were recorded on a BIAcoreX. The bars at 0 and 300 sec indicate the

start of injection of ligand (association phase) and the start of injection of buffer alone

(dissociation phase), respectively. Concentrations of Ngb used were 1, 2, 4, 6, 8 and 10 µM. B,

SPR analyses of ferric or ferrous-CO Ngb binding to Gαi1. The concentrations of Ngbs were 4

µM. C, Effects of a guanine nucleotide (GDP, or GDP plus AlF4-) on the interaction between Ngb

and Gαi1. The concentration of Ngb was 3 µM.

FIG. 3. Effects of human Ngb on GTPγS binding to Gαi/o. The binding of GTPγS to 100 nM

Gαi1 (A) and Gαo (B) in the absence (○) or presence of 5 µM ferric (●) or ferrous-CO Ngb (▲)

was initiated by the addition of 50 nM [35S]GTPγS (>1000 Ci/mmol). Gαi1- or Gαo-bound

GTPγS was counted by withdrawing aliquots at the indicated times and passing through

nitrocellulose filters (0.45 µm).

FIG. 4. Effects of Ngb on dissociation of GDP from GDP-bound Gαi1. A, Experiments

performed in the presence of an excess amount of unlabeled GTP. Gαi1 complexed with [3H]GDP

was obtained as described in the “Materials and methods”. An excess of unlabeled GTP (200 µM)

was added to the Gαi1:[3H]GDP complex in the absence or presence of 5 µM Ngb. Aliquots were

withdrawn at 0, 5, and 10 min and passed through nitrocellulose filters (0.45 µm). Each error bar

represents the standard deviation of 3-4 independent experiments. B, Experiments performed in

the presence of an excess amount of unlabeled GDP. Experimental conditions were as in A,

except for the addition of an excess amount of unlabeled GDP (200 µM).

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

27

FIG. 5. SDS-PAGE analyses of human Ngbs under nonreducing conditions. A, analysis of

human wild-type Ngb. Human wild-type Ngb was incubated in the absence or presence of 2 mM

DTT for 2 hours at 25 °C. The samples were analyzed under nonreducing conditions on 12.5 %

SDS-polyacrylamide gels and stained by Coomassie Blue. Molecular size markers are given to

the left (in kilodaltons). B, analysis of human Ngb mutants [triple mutant (C46S,C55S,C120S)

and double mutant (C55S,C120S)] with C-terminal 6xHis tag under nonreducing conditions.

Experimental conditions are as in A.

FIG. 6. Effects of disulfide bond formation on GDI activities of ferric Ngbs. A, Experiments

performed in the presence of an excess amount of unlabeled GTP. Human wild-type Ngb and

Ngb mutants [triple mutant (C46S,C55S,C120S) and double mutant (C55S,C120S) with C-

terminal 6xHis tag] were used. Percentages of [3H]GDP-bound Gαi1 at 5 min are shown. The

concentrations of Ngbs were 5 µM. Experimental conditions are as in Fig. 4A. B, Experiments

performed in the presence of an excess amount of unlabeled GDP. Experimental conditions are as

in Fig. 4B.

FIG. 7. Human Ngb as an oxidative-stress responsive sensor for signal transduction in the

brain.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

DE

F

FG

H

α3α4

α5

α6α7

α8

Mb 42 EKFDKFK-HLKSEDEMK--ASEDLKKHGATVLTALGGILKKKGH---HEAEIK 88

Ngb 40 PLFQYNCRQFSSPEDCL--SSPEFLDHIRKVMLVIDAAVTNVEDLSSLEEYLA 90

GRK2 62 KLGYLLFRDFCLNHLEEARPLVEFYEEIKKYEKLETEEERVARSREIFDSYIM 114

GRK4 61 PIGRRLFRQFCDTKPTLK-RHIEFLDAVAEYEVADD-EDRSDCGLSILDRFFN 111

GRK5 61 PIGRLLFRQFCETRPGLE-CYIQFLDSVAEYEVTPD-EKLGEKGKEIMTKYLT 111

GRK6 61 PIGRLLFREFCATRPELS-RCVAFLDGVAEYEVTPD-DKRKACGRQLTQNFLS 111

RGS1 80 QTGQNVFGSFLKSEFSE--ENIEFWLACEDYKKTES-DLLPCKAEEIYKAFVH 129

RGS4 70 ECGLAAFKAFLKSEYSE--ENIDFWISCEEYKKIKSPSKLSPKAKKIYNEFIS 120

RGS9 81 PKGRQSFQYFLKKEFSG--ENLGFWEACEDLKYGDQ-SKVKEKAEEIYKLFLA 130

GAIP 98 PAGRSVFRAFLRTEYSE--ENMLFWLACEELKAEANQHVVDEKARLIYEDYVS 148

Mb 89 PLAQSHATKHKIPVKYLEFISEAIIQVLQS-KHPGDFGADAQGAMNKALELFRK 141

Ngb 91 SLGRKHRAVG-VKLSSFSTVGESLLYMLEK-CLGPAFTPATRAAWSQLYGAVVQ 142

GRK2 115 KELLAC-SHP-F---SKSATEHVQGHLGKKQVPPDLFQPYIEEICQNLRGDVFQ 163

GRK4 112 DKLAAP-LPE-I---PPDVVTECRLGLKEENPSKKAFEECTRVAHNYLRGEPFE 160

GRK5 112 PKSPVF-IAQ-V---GQDLVSQTEEKLLQK-PCKELFSACAQSVHEYLRGEPFH 159

GRK6 112 HTGPDL-IPE-V---PRQLVTNCTQRLEQG-PCKDLFQELTRLTHEYLSVAPFA 159

RGS1 130 SDAAK--QIN-I---DFRTRESTAKKIKA--PTPTCFDEAQKVIYTLMEKDSYP 175

RGS4 121 VQATK--EVN-L---DSCTREETSRNMLE--PTITCFDEAQKKIFNLMEKDSYR 166

RGS9 131 PGARR--WIN-I---DGKTMDITVKGLKH--PHRYVLDAAQTHIYMLMKKDSYA 176

GAIP 149 ILSPK--EVS-L---DSRVREGINKKMQE--PSAHTFDDAQLQIYTLMHRDSYP 194

Fig

. 1

by guest on March 22, 2018http://www.jbc.org/Downloaded from

0

50

100

150

200

250

300

350

400

0 200 400 600 800

SP

R S

igna

l (R

U)

Time (sec)

10

8

6

4

2

1

Fig. 2A

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

50

100

150

200

0 200 400 600 800

SP

R S

igna

l (R

U)

Time (sec)

Ferrous-CO Ngb

Ferric Ngb

Fig. 2B

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

-10

0

10

20

30

40

50

60

0 200 400 600 800

SP

R S

igna

l (R

U)

Time (sec)

+ GDP

+ GDP + AlF4-

Fig. 2C

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

20

40

60

80

100

0 5 10 15 20

GT

P

S b

indi

ng (

% m

axim

um)

Time (min)

γ

Fig. 3A

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

10

20

30

40

50

60

70

0 5 10 15 20

GT

P

S b

indi

ng (

% m

axim

um)

Time (min)

γ

Fig. 3B

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

20

40

60

80

100

120G

DP

bou

nd (

% m

axim

um)

Gα i1 +NgbGα i1

0 5 10 0 5 10

Fig. 4A

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

20

40

60

80

100

120G

DP

bou

nd (

% m

axim

um)

Gα i1 +NgbGα i1

0 5 10 0 5 10

Fig. 4B

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

DTT -+ +

47.0

38.0

29.5

21.0

9.8

Fig. 5A

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

DTT - + +-

47.0

38.0

29.5

21.0

Triple mutant(C46S,C55S,C120S)

Double mutant (C55S,C120S)

Fig. 5B

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

20

40

60

80

100

120G

DP

bou

nd (

% m

axim

um)

Triplemutant+ DTT

Doublemutant+ DTT

Triplemutant- DTT

Doublemutant- DTT

WT- DTT

WT+ DTT

Fig. 6A

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

20

40

60

80

100

120G

DP

bou

nd (

% m

axim

um)

Triplemutant+ DTT

Doublemutant+ DTT

Triplemutant- DTT

Doublemutant- DTT

WT- DTT

WT+ DTT

Fig. 6B

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

GDP-boundGα

NormoxiaHypoxia

(Ischemia, Reperfusion)

oxidative stressNo signal

GTP-boundGα GβγGDP-bound

Gα

Gβγ

GTPGDP

inactive active

Effectors Effectors

GPCR

Ferrous-02 Ngb

Ferric Ngb

Gβγ

inactive active

Effectors

Cell survival

?

Ferric Ngb

Fig. 7

Pi

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

FIGURE LEGEND

Supplement. SPR analyses of Mb or Gββββγγγγ binding to G protein αααα-subunit Gααααi1. Gαi1

was immobilized to a CM5 sensor chip. The on and off processes for ligand binding

were recorded on a BIAcoreX. The bars at 0 and 300 sec indicate the start of injection

of ligand (association phase) and the start of injection of buffer alone (dissociation

phase), respectively. As a negative control, human Mb (10 µM) was used. Human Mb

did not bind to Gαi1 (Kd > 1 mM). As a positive control, concentration dependence of

Gβγ on binding affinities with Gαi1 was investigated. Concentrations of Gβγ used were

3, 9 and 25 nM. The running buffer containing 5 mM MgSO4 and 500 µM GDP was

used. Equilibrium dissociation constant for the interaction of Gβγ with Gαi1 Kd = 6.7

nM.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

50

100

150

200

250

0 200 400 600 800

SP

R S

igna

l (R

U)

Time (sec)

Gβγ 25 nM

Gβγ 9 nM

Gβγ 3 nM

Mb 10 Mµ

Supplement

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Keisuke Wakasugi, Tomomi Nakano and Isao Morishimanucleotide dissociation inhibitor

Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine

published online July 14, 2003J. Biol. Chem. 

  10.1074/jbc.M305519200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2003/07/23/M305519200.DC1

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from