Combining data from genomes, Y2H and 3D structure indicates that BolA is a reductase interacting with a glutaredoxin.

Abstract

Genomes, functional genomics data and 3D structure reflect different aspects of protein function. Here, we combine these data to predict that BolA, a widely distributed protein family with unknown function, is a reductase that interacts with a glutaredoxin. Comparisons at the 3D structure level as well as at the sequence profile level indicate homology between BolA and OsmC, an enzyme that reduces organic peroxides. Complementary to this, comparative analyses of genomes and genomics data provide strong evidence of an interaction between BolA and the mono-thiol glutaredoxin family. The interaction between BolA and a mono-thiol glutaredoxin is of particular interest because BolA does not, in contrast to its homolog OsmC, have evolutionarily conserved cysteines to provide it with reducing equivalents. We propose that BolA uses the mono-thiol glutaredoxin as the source for these.

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Hypothesis
Combining data from genomes, Y2H and 3D structure indicates
that BolA is a reductase interacting with a glutaredoxin
Martijn A. Huynena,b,*, Chris A.E.M. Spronkb, Toni Gabaldo ´na,b, Berend Snela,b
aCMBI, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Toernooiveld 1,
6525ED Nijmegen, The Netherlands
bCenter for Molecular and Biomolecular Informatics, Radboud University Nijmegen, Toernooiveld 1, 6525ED Nijmegen, The Netherlands
Received 15 September 2004; revised 22 October 2004; accepted 9 November 2004
Available online 29 December 2004
Edited by Robert B. Russell
Abstract
reflect different aspects of protein function. Here, we combine
these data to predict that BolA, a widely distributed protein fam-
ily with unknown function, is a reductase that interacts with a
glutaredoxin. Comparisons at the 3D structure level as well as
at the sequence profile level indicate homology between BolA
and OsmC, an enzyme that reduces organic peroxides. Comple-
mentary to this, comparative analyses of genomes and genomics
data provide strong evidence of an interaction between BolA and
the mono-thiol glutaredoxin family. The interaction between
BolA and a mono-thiol glutaredoxin is of particular interest be-
cause BolA does not, in contrast to its homolog OsmC, have evo-
lutionarily conserved cysteines to provide it with reducing
equivalents. We propose that BolA uses the mono-thiol glutare-
doxin as the source for these.
? ? 2004 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Genomes, functional genomics data and 3D structure
Keywords: BolA; Comparative genomics; Mono-thiol
glutaredoxin; PICOT-HD; Protein function prediction;
Uvi31+
1. Introduction
In the genomics era, we obtain many correlates of protein
function, such as a protein?s 3D structure, its gene expression,
its physical interaction partners, the location of its gene on the
genome and its phylogenetic distribution. The various sources
of information are stored in dedicated databases that provide
invaluable resources for the prediction of the biological func-
tion of a protein. Nevertheless, none of these give a direct an-
swer to the question: what does the protein do at the molecular
level? However, by combining on the one hand homology
information, that can be deduced from 3D structure compari-
sons and that provides information about the molecular func-
tion of a protein, with on the other hand genomics context
data that provide information about the interaction partners
or substrates of a protein, one can derive specific hypotheses
about its function.
Here, we combine genome sequences, physical interaction
data and 3D structures to provide a specific prediction for
the function of BolA, a protein family that is widespread
among proteobacteria and eukaryotes including Homo sapiens.
Despite a considerable amount of research on the function of
BolA, its molecular function remains unknown. Originally,
bolA been identified as a gene that causes round morphology
in Escherichia coli when overexpressed [1]. No phenotype for
strains lacking bolA has been found for cells growing on a rich
medium. Consistent with this is that bolA is mainly expressed
under the stress conditions like the stationary phase, osmotic
shock, carbon starvation and oxidative stress [2]. The gene
bolA is under control of RpoS, which also regulates the expres-
sion of other stationary-phase-induced stress genes [3]. A
homolog of bolA in Schizosaccharomyces pombe, uvi31+, is ex-
pressed under UV radiation [4], which is known to stimulate
the intracellular synthesis of reactive oxygen species. Although
no direct evidence about BolA?s function is available, its asso-
ciated phenotypes link BolA to cell morphology and cell divi-
sion. Under nutrient-restrictive conditions bolA in E. coli is
required for normal cell morphology [2], while uvi31+ in S.
pombe has been implicated in the regulation of septation and
cytokinesis [5]. How the link between BolA and cell division
and morphology is effectuated is however not clear. Overex-
pression of bolA in E. coli leads to the upregulation of cell wall
synthesis genes dacA (PBP5), dacC (PBP6) and ampC (AmpC),
and BolA?s effect on the cell morphology appears dependent
on PBP5 and PBP6 [6]. Bola has therewith been proposed to
be a regulator of cell wall biosynthetic enzymes [6]. Recently,
the structure of the BolA homolog in Mus musculus has been
determined [7]. BolA has a class II KH fold, instances of which
are known to bind DNA and RNA and therewith support a
regulatory role for BolA.
Here, we show that the accumulating wealth of genomics
data indicates however a different molecular function for
BolA. We show that BolA is homologous to the peroxide
reductase OsmC and, relative to other class II KH-fold pro-
teins, most closely related to it, and that BolA has a very
strong genomic association with the mono-thiol glutaredox-
ins/PICOT-HD [8] family. These data point to a role of BolA
as a reductase that functions in conjunction with a mono-thiol
glutaredoxin. The thiol group of the latter would potentially be
used by BolA to reduce and/or deglutathionylate substrates.
*Corresponding author. Fax: +31-24-3652977.
E-mail addresses: huynen@cmbi.ru.nl (M.A. Huynen),
c.spronk@cmbi.ru.nl (C.A.E.M. Spronk), t.gabaldon@cmbi.ru.nl
(T. Gabaldo ´n), snel@cmbi.ru.nl (B. Snel).
0014-5793/$30.00 ? 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2004.11.111
FEBS 29201 FEBS Letters 579 (2005) 591–596

Page 2

2. Methods
The phylogeny of the BolA family (Fig. 1) was reconstructed with
PhyML [9] based on a sequence alignment created with muscle [10],
the sequence identifiers are: Q9VC53 Q9D8S9 YCE3_HUMAN
YN1I_CAEEL Q92ME9 Q84W65 Q9ZDT0 UV31_SCHPO Q87BI5
Q8XYL3 BOLA_ECOLI Q7VKT0 Q88M18 ENSCBRP00000005226
Q8CEI1Q9USK1YAE6_YEAST
MY16_MOUSEQ8ML41 YGX0_YEAST
Q9XVJ0 Q98NN9 Q9HQ28 Q7P9Z5 YRBA_ECOLI YA82_HAEIN
Q9HVW6 Q8DKI4 Q87DN9 Q8F4D1 Q8XV77 Q9LF68.
Sequence alignments for the comparison of BolA with the other
class II KH-fold families were obtained from PFAM [11]. The E values
for the profile comparison were calculated with Compass, the database
size used (-d option in compass) was the combined length of all the
KH-fold alignments.
Q8F4C9MY16_HUMAN
O14280 Q9FIC3
3. Results
3.1. BolA is homologous to the peroxide reductase OsmC
Comparative sequence analyses indicate that homologs of
bolA are present in Bacterial and Eukaryotic genomes. Consis-
tent with earlier findings from large-scale phylogenetic analyses
[12], the eukaryotic representatives of bolA appear derived from
the proteobacteria (Fig. 1) and presumably have hitchhiked
along with the endosymbiosis of an a-proteobacterium that be-
came the mitochondrion. The molecular function of BolA is
unknown and using sequence-to-profile searches (PSI-Blast)
[13], we could not detect homology of BolA to sequences with
a known molecular function. Recently, however, the 3D struc-
ture of a Mus musculus member of the BolA family has been
published [7]. According to the Dali 3D structure classification
system, BolA is homologous to OsmC (Table 1). BolA and the
N-terminal domain of OsmC have a class II KH fold. They dis-
tinguish themselves from other members of this fold because
they both miss the GxxG element [7], which otherwise is well
conserved in the class II KH fold and which appears essential
Fig. 1. Phylogeny of the BolA family with interaction predicting genomics data. Different types of genomic contexts are indicated, ‘‘GRX’’ indicates
the mono-thiol glutaredoxin family. Physical interaction between BolA and a mono-thiol glutaredoxin is supported by various types of genomics
data across a wide phylogenetic range.
Table 1
Similarity of BolA to other class II KH-fold proteins
Protein family
(PDB entry)
3D similarity to
Bola, Z scores
Sequence profile
similarity to Bola.
SW score (E value)
OsmC (1ml8A/1lqlA)
Ohr (1n2fA)
5.8/5.5
5.2
73 (2.4E ? 5)
KH 1 (1hnxC)
DUF150 (1ib8A)
GMP synthase C (1gpmA)
KH 2 (1egaB)
RBFA (1kkgA)
5.3
3.7
2.9
3.8
4.2
46 (9.4E ? 3)
44 (4.2E ? 2)
57 (7.0E ? 4)
35 (2.7E ? 1)
40 (9.6E ? 2)
The similarities of the BolA structure to other protein structures of the
class II KH fold are indicated at the 3D structure level as well as at the
sequence profile level. The Z scores are calculated with DALI [35],
using pairwise comparisons of the BolA structure with other class II
KH folds in the DALI database. The higher the Z score, the less likely
it is that the similarity between the 3D structures is ‘‘random’’. The
Smith–Waterman scores and E values for the comparison of sequence
profiles were calculated with Compass [15], the higher the score, the
more similar the sequence profiles. For the profile similarity analysis,
one combined alignment was used of the OsmC and Ohr subfamilies,
as they form one protein family. Both structure comparison as well as
sequence profile comparison clearly indicate that BolA is homologous
to the OsmC/Ohr family as well as most similar to the OsmC/Ohr
family relative to other members of the class II KH fold.
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for nucleic acid binding [14]. In terms of 3D structure similarity,
BolA is most similar to OsmC (Table 1), relative to other pro-
teins with a class II KH fold and the structures can readily be
superimposed (Fig. 2). The main difference between the BolA
and OsmC structures is that the a3-helix of BolA is one turn
shorter than the corresponding helix of OsmC (Fig. 2). Com-
pass-based comparisons [15] of the sequence profile of BolA
with the profiles of the other members of the class II KH fold
indicate that also at the sequence level BolA is most similar
to the OsmC family that also includes the thiol-dependent
reductase Ohr (Table 1). OsmC is a hyperperoxide reductase
that can detoxify hydroperoxides by reducing them into alco-
hols [16]. The active site of OsmC has two conserved cysteines,
which have been proposed to carry the reducing equivalents for
OsmCs reductive activity [16]. These cysteines are however not
conserved in BolA. If BolA functions as a reductase, as the
Fig. 2. Superposition of the 3D structures of BolA (red) and OsmC
(green). The structures, BolA of M. musculus (1v9j) and OsmC of E.
coli (1ml8), were aligned with SHEBA [36]. Indicated are the a1, a2, a3
helix and the 310helix of BolA, the nomenclature is adapted from
Kasai et al. [7]. The main difference between the structures is that the
a3 helix of OsmC is one turn longer than the one of BolA.
Fig. 3. Phylogenetic distribution of BolA and of the mono-thiol glutaredoxin family (Grx). Data were obtained with STRING [18]. Among 145
sequenced genomes, the phylogenetic distribution of the genes is virtually identical. The only exception is Encephalotozoon cuniculi. Wigglesworthia
brevipalpis does not contain bolA in the COG orthology database [37] that is used in STRING, it does however contain a homolog of bolA, which in
COGs has been classified as bolA?s paralog yrbA.
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homology with OsmC indicates, it has to obtain its reducing
equivalents from another source.
3.2. Gene order conservation and gene co-occurrence indicate an
interaction of BolA with a mono-thiol glutaredoxin
Potential interaction partners for BolA, which could provide
these reducing equivalents, can be found by comparative gen-
ome analysis. For bolA, we have analyzed a number of so-
called ‘‘genomic context’’ [17] types (gene fusion, gene-order
conservation, and the co-occurrence of genes among se-
quenced genomes) using the genomic context server STRING
[18]. Two types of genomic context indicate an interaction be-
tween BolA and a mono-thiol glutaredoxin/PICOT-homology
domain [8]: Their conserved occurrence as neighbors in Bacte-
rial genomes (Fig. 1) and the co-occurrence of their genes
across virtually all sequenced genomes (Fig. 3). Either type
of genomic context has been successfully used to predict func-
tional interactions in the past [19] and their combined presence
leads to an estimated likelihood of interaction of BolA with a
mono-thiol glutaredoxin of 97% [18]. In general, genomic con-
text data do not indicate what type of interaction (metabolic,
physical or regulatory) two proteins have, although when, as
in this case, the association is very strong, the interaction does
tend to be a physical one [17].
3.3. An evolutionary conserved physical interaction of BolA with
Grx3
We examined genomics databases of physical interaction
experiments to establish whether BolA has indeed a physical
interaction with a mono-thiol glutaredoxin. In S. cerevisiae,
both in the yeast-2-hybrid experiments [20] as well in the
FLAG tag experiments [21], an interaction of Grx3 (a S. cere-
visiae homolog of the bacterial mono-thiol glutaredoxins) with
YGL220w (a S. cerevisiae BolA homolog) has been detected.
The detection of such an interaction with two independent
methods increases the likelihood that they are indeed biologi-
cally relevant [22]. Furthermore, this interaction has also been
observed in another species. In a yeast-2-hybrid assay on Dro-
sophila melanogaster, CG16804 and CG6523, the orthologs of
YGL220w and Grx3, respectively, have also been shown to
interact with each other [23]. Such evolutionary conservation
of a yeast-2-hybrid interaction increases the likelihood of bio-
logical relevance to 100% [24,25]. A physical interaction of
BolA with a mono-thiol glutaredoxin thus appears to be more
than likely.
3.4. The mono-thiol glutaredoxin/PICOT-HD family
Glutaredoxins are redox enzymes that use glutathione to
catalyze disulfide reductions [26]. Consistent with their interac-
tion with BolA is that glutaredoxins are, like BolA, involved in
oxidative stress response [26]. Within the glutaredoxins, the
mono-thiol glutaredoxins or PICOT-HD [8] form a separate
group that lack one of the two conserved cysteines of the dith-
iol glutaredoxins, and that are frequently fused with thioredox-
ins in eukaryotes [8]. Their exact function has not been
elucidated. It has been suggested that they are involved in
the deglutathionylation of protein-GS mixed sulfides [27,28].
For this process dithiol glutaredoxins only require their N-ter-
minal cysteine thiol [29], which is the one cysteine that is con-
served inmono-thiolglutaredoxins.
characterized member of this family, the mitochondrial protein
Indeedthe best
Grx5 from yeast, is able to deglutathionylate proteins [27], and
is involved in defense against oxidative stress [30].
3.5. Interpreting the interaction between BolA and Grx3
Given the strong link between BolA and Grx3, they likely
function as a complex and are both involved in the same pro-
cess. BolA could, e.g., use the reducing equivalents from Grx3
to reduce and/or deglutathionylate some substrate. In that
case, the reducing equivalents from Grx3 would ‘‘replace’’
the ones that are carried by the conserved cysteines from
OsmC and that are absent from BolA. An alternative is that
one of the proteins is a target for the other and the interaction
is ‘‘transient’’. In that case, BolA could play a role in the deglu-
tathionylation of Grx3 itself. Here, it should be noted that glu-
tathione is able to deglutathionylate Grx5, albeit at a 20-fold
lower rate than for the reduction of dithiolic glutaredoxin from
E. coli [27].
3.6. The link with cell division
Genomic context data do also indicate other interaction
partners of BolA (Fig. 4). Given BolA?s phenotypic links to
cell division, most interesting are conservation of gene order
with the gene for intracellular septation protein A (ISPA)
[31] and with the gene for MurA that catalyzes the first step
of peptidoglycan synthesis. Neither of these proteins can how-
ever be detected in eukaryotes. A link to the cell wall that does
carry to the eukaryotes is with a Parvulin-like peptidyl–prolyl
isomerase D (PPIase D) whose gene has a conserved gene or-
der with bolA in prokaryotes, and is co-expressed with it in S.
cerevisiae [18]. PPIase D is, in E. coli, a periplasmic chaperone
that is required for folding of outer membrane proteins [32].
Finally, there is conservation of gene order with an ABC-
transporter system that is involved in tolerance to toluene
[33], and that has been predicted to be an efflux system [33],
and there is gene co-occurrence with Glutathione-S-transfer-
Fig. 4. Predicted interactions of the BolA family. The various types of
data that support the interactions are indicated. The proteins on the
left can be linked to the cell wall or cell division: MurA is involved in
cell wall synthesis, IspA is involved in cell division, and PPIase D is a
periplasmic chaperone. The proteins on the right are more generally
involved in (oxidative) stress response: Ttg1, Ttg2 and Ttg3 form an
ABC transporter involved in resistance to Toluene, GST is Glutathi-
one-S-transferase, and Grx is mono-thiol glutaredoxin. The results
were obtained with STRING [18]. In the figure, the results for BolA/
COG0271 and for its paralog YrbA/COG5007 are combined because
the COG subclassification of the BolA family does not match the
phylogenetic tree (data not shown) and therefore appears unreliable.
594
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Page 5

ase, that functions in defense against oxidative stress. The links
with MurA, IspA and PPIase D, although significant in them-
selves and consistent with the phenotypic data on bolA, are not
as strongly supported by any type of data, nor are they sup-
ported by so many types of data as the link between BolA
and the mono-thiol glutaredoxins. They suggest that the tar-
gets of the BolA-glutaredoxin pair are proteins or other organ-
ic compounds that are either part of the membrane or cell wall
or that are involved in its generation.
4. Discussion
Protein function predictions have been made based on simi-
larity at the level of the sequence or 3D structure (reviewed in
[34]), or on combinations of homology with either functional
genomics data, e.g. [23] or genomic context data [17]. Here we
combine 3D structure data with both genomic context data
and functional genomics data. The increasing pace at which
all these types of data are becoming available for hypothetical
proteins calls for a further integration of genomic context dat-
abases like STRING [18] with homology databases like DALI
[35] to facilitate the makingof specific hypotheses about protein
function.Thechallengewilltherebyincludefunctionalinforma-
tion that assists in observing a potential link between ‘‘BolA is
homologous to a peroxide reductase’’ and ‘‘BolA interacts with
a mono-thiol glutaredoxin’’. Such a level of integration would
be of tremendous value for reaping the benefits of genomics
for the understanding of the cell at the molecular level.
Acknowledgment: We thank Sander Nabuurs for assistance with the
protein structure alignment. This work was supported in part by a
grant from the Netherlands Organization for Scientific Research
(NWO).
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