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LysM, a widely distributed protein motif for binding to (peptido)glycans
Buist, Girbe; Steen, Anton; Kok, Jan; Kuipers, Oscar
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Molecular Microbiology
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10.1111/j.1365-2958.2008.06211.x
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Buist, G., Steen, A., Kok, J., & Kuipers, O. P. (2008). LysM, a widely distributed protein motif for binding to
(peptido)glycans. Molecular Microbiology, 68(4), 838-847. DOI: 10.1111/j.1365-2958.2008.06211.x
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Molecular Microbiology (2008) 68(4), 838–847 䊏
doi:10.1111/j.1365-2958.2008.06211.x
First published online 8 April 2008
MicroReview
LysM, a widely distributed protein motif for binding
to (peptido)glycans
Girbe Buist,†§ Anton Steen,‡§ Jan Kok and
Oscar P. Kuipers*
Department of Molecular Genetics, Groningen
Biomolecular Sciences and Biotechnology institute,
University of Groningen, Kerklaan 30, 9751 NN Haren,
the Netherlands.
Summary
Bacteria retain certain proteins at their cell envelopes
by attaching them in a non-covalent manner to peptidoglycan, using specific protein domains, such as
the prominent LysM (Lysin Motif) domain. More than
4000 (Pfam PF01476) proteins of both prokaryotes
and eukaryotes have been found to contain one or
more Lysin Motifs. Notably, this collection contains
not only truly secreted proteins, but also (outer)membrane proteins, lipoproteins or proteins bound
to the cell wall in a (non-)covalent manner. The motif
typically ranges in length from 44 to 65 amino acid
residues and binds to various types of peptidoglycan and chitin, most likely recognizing the
N-acetylglucosamine moiety. Most bacterial LysMcontaining proteins are peptidoglycan hydrolases
with various cleavage specificities. Binding of certain
LysM proteins to cells of Gram-positive bacteria has
been shown to occur at specific sites, as binding
elsewhere is hindered by the presence of other cell
wall components such as lipoteichoic acids. Interestingly, LysM domains of certain plant kinases enable
the plant to recognize its symbiotic bacteria or sense
and induce resistance against fungi. This interaction
is triggered by chitin-like compounds that are
secreted by the symbiotic bacteria or released from
Accepted 8 March, 2008. *For correspondence. E-mail o.p.kuipers@
rug.nl; Tel. (+31) 50 3632093; Fax (+31) 50 3632 348. Present
address: †Laboratory of Molecular Bacteriology, Department of
Medical Microbiology, University Medical Center Groningen, Hanzeplein 1, 9700 RB, Groningen, the Netherlands; ‡Membrane Enzymology Groningen Biomolecular Sciences and Biotechnology
institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen,
the Netherlands. §These authors contributed equally.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
fungi, demonstrating an important sensing function
of LysMs.
The Lysin Motif
The Lysin Motif (LysM) was first discovered in the
lysozyme of Bacillus phage f29, where it was detected as
a C-terminal direct repeat composed of 44 amino acids
(aa) separated by 7 aa (Garvey et al., 1986). Similar
motifs were subsequently observed in the peptidoglycan
(PG) hydrolase of Enterococcus faecalis (Béliveau et al.,
1991). This enzyme contains a C-terminal LysM domain
with six LysMs for which a PG binding function was
postulated. Currently, various proteins containing LysMs
have been studied (Table S1). LysMs occur frequently in
bacterial lysins, in bacteriophage proteins and in certain
proteins of eukaryotes (Pfam PF01476 and Prodom
PD407905). They are also present in bacterial PG hydrolases and in peptidases, chitinases, esterases, reductases or nucleotidases. They can act as antigens or are
proteins that bind, for example, to albumin, elastin or
immunoglobulin (Desvaux et al., 2006). Up to now, LysMs
have not been found in archaeal proteins (Fig. 1). Multiple
LysMs within one LysM domain are separated by spacing
sequences mostly consisting of Ser, Thr and Asp or Pro
residues (Buist et al., 1995; Ohnuma et al., 2008), which
may form a flexible region between the LysMs. The intervening sequences vary in length, composition and do not
share significant homology. However, the intervening
sequences between the LysMs of a family of receptor-like
kinases in plants contain a conserved CxC motif, for
which the function is unknown (Madsen et al., 2003;
Radutoiu et al., 2003; Arrighi et al., 2006). LysMs are
present in the N-terminal as well as the C-terminal
domains of proteins; they are also present in the central
part of proteins, possibly connecting two (catalytic)
domains (Table S1).
The consensus sequence of all available LysMs (Fig. 2)
shows that the motif is well conserved over the first 16 and
somewhat less over the last 10 amino acid residues. The
central region is poorly conserved except for the Ile/Leu
at positions 23 and 30 and the well-conserved Asn at
LysM, a widely distributed glycan binding domain 839
Fig. 1. Taxonomic coverage of the LysM
domain. Picture was generated by the interpro
website (http://www.ebi.ac.uk/interpro/
IEntry?ac=IPR002482). The root of the
taxonomy tree is placed at the centre of the
circles. The outer circle contains some
selected model organisms, with the nodes of
the tree leading to these model organisms on
the inner circles. The position of the nodes
on the inner circles is chosen for
convenience.
position 27. Prokaryotic LysMs, in contrast to their eukaryotic counterparts, do not possess possible disulphide
bridges. Their domains contain extensive secondary
structures and hydrogen bond networks, and consequently, disulphide bridges are non-essential for their
structure and functions (Ponting et al., 1999). Interestingly, the isoelectric points (pIs) of the LysM proteins
range from 4 to 12, with most having a pI of 5 or 10
(Table S1). In some cases, one organism expresses two
paralogous LysM domain-containing proteins with different pIs; for example, the homologous PG hydrolases
AcmA and AcmD of Lactococcus lactis contain three
C-terminal LysMs with pIs of around 10 and 4
respectively. The exact function of LysM domains with low
pI is unknown, but they are observed in proteins of several
bacteria and could be a way to adapt to changing pHs in
the environment or to display an altered substrate binding
specificity.
N-acetylglucosamine is the general constituent in binding
substrates of LysM proteins
The best-characterized LysM-containing protein is the
N-acetylglucosaminidase AcmA of L. lactis (Buist et al.,
1995). AcmA binds in a non-covalent manner to the cell
wall and is responsible for cell lysis of producing cells as
well as lysis in trans (Fig. 3) (Buist et al., 1997). A fusion
of a Malaria parasite surface antigen and the C-terminal
LysM domain of AcmA bound specifically to cell walls of
AcmA-producing and AcmA-non-producing L. lactis cells
and to whole cells of several other Gram-positive bacterial species tested (Steen et al., 2003). Binding was
obtained in the range of pH 4–10. A similar fusion with the
LysM domain of AcmD only bound at a pH below the pI of
that domain (~pH 4) (G. Buist et al., unpublished), suggesting that positive charges play an important role in
binding. Binding studies using the AcmA fusion protein
and chemically treated L. lactis cells and cell walls identified PG as the component to which the LysM domain of
AcmA binds (Steen et al., 2003). Binding to purified PG
has also been shown for AtlA of E. faecalis, a PG hydrolase containing six C-terminal LysMs (Eckert et al., 2006).
The LysM domain of AcmA has similar affinity for both
A-type and B-type PG (Steen et al., 2003). As repetition
of the disaccharide N-acetylglucosamine (GlcNAc) and
N-acetylmuramic acid (MurNAc) is the only common part
in A- and B-type PG, LysM domains most likely bind to
this component of the PG. No binding to (GlcNAc)3 was
obtained with the LysM domain of AcmA (A. Steen et al.,
unpublished).
Fig. 2. Consensus sequence of LysM (Pfam database entry PF01476) (adapted from Desvaux et al., 2006). Above the sequence the location
and length (N = number of amino acid residues) of the inserts among the LysMs are indicated. The consensus residues of the YG domain
(YXXXXGXXHy, Turner et al., 2004) that are also present in the GW domain (Prodom entry PD006903) and the Cholin domain (PD000439)
are boxed. Boxes below the sequence indicate regions with secondary structure as defined by Mulder et al. (2006).
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 838–847
840 G. Buist, A. Steen, J. Kok and O. P. Kuipers 䊏
Fig. 3. Schematic presentation of various predicted cellular locations of investigated LysM-containing proteins. OM, outer membrane;
IM, inner membrane; OWZ, outer wall zone; IWZ, inner wall zone; OC, outer cortex; IC, inner cortex; LysM motifs, open ovals. Details of the
proteins indicated are listed in Table S1.
Members of the type III sugar binding antiviral protein
CyanoVirin-N Homology (CVNH) contain one LysM. The
CVN domain is a small 11 kDa domain that binds to the
mannose moieties of surface glycoproteins of viruses like
HIV and Ebola, thereby blocking entry of the virus into the
host (Percudani et al., 2005). Type III proteins of the CVN
homology family are only present in filamentous ascomycetes and contain two CVNH domains separated by
a LysM. Interestingly, b1,4-N-acetylglucosamine is the
reducing end of the Man9 oligosaccharide moiety associated with the viral surface glycoproteins gp120.
In some eukaryotes, e.g. in the nematode Caenorhabditis elegans and in the green alga Volvox carteri, LysMs
are present in chitinases (Bateman and Bycroft, 2000),
suggesting that they may bind to chitin, a polymer of
GlcNAc that resembles the glycan chain of PG. The striking sequence differences between LysMs of chitinases
and those of the bacterial cell wall-hydrolysing enzymes,
in particular the presence of multiple Cys residues in the
former, could account for the difference in binding
specificity. The Nod factors secreted by Rhizobium
species, recognized by the LysM domains of plant receptors, consist of a GlcNAc backbone of four or five residues
(Radutoiu et al., 2007). Chitin-like compounds are not
only involved in the signalling between bacteria and
plants, they are also involved in inducing plant defence
responses against fungi, which have chitin as the major
component of their cell walls. In Arabidopsis and rice the
defence response against fungi is induced via the LysMcontaining receptor-like kinases CERK1, LysM RLK1 and
CEBiP respectively (Kaku et al., 2006; Miya et al., 2007;
Wan et al., 2008).
Recently is has been proven that the two N-terminal
LysMs of the chitinase from Pteris ryukyuensis bind
(GlcNAc)5 with a stoichiometry of 1:1 (Ohnuma et al.,
2008). Poor binding was obtained with (GlcNAc)3 while an
increasingly stronger binding was obtained with (GlcNAc)4
and (GlcNAc)5.
Together these data show that GlcNAc is the common
sugar bound by LysMs, but binding studies using LysMs
from different proteins and a variety of possible substrates
are needed to determine whether besides GlcNAc,
MurNAc, for example, possibly together with some of the
peptide stem residues in PG, is also recognized by LysMs.
Structure of the Lysin motif
The structure of the LysM of the Escherichia coli outer
membrane-bound lytic murein transglycosylase MltD has
been solved by NMR (Bateman and Bycroft, 2000). The
LysM has a baab secondary structure with the two
a-helices packing onto the same side of an antiparallel
b-sheet. Using this information, the structure of the
CVNH-LysM protein from a filamentous ascomycete was
predicted (Percudani et al., 2005). The crystal structure of
the Bacillus subtilis spore protein YkuD, which contains
one N-terminally located LysM, was solved by multiwavelength anomalous dispersion (Bielnicki et al., 2006).
The structure of the LysM in YkuD was similar to that of
MltD. Two glycin residues (residues 6 and 38 in Fig. 2) are
highly conserved in LysMs and are part of tight turns in the
LysM structure. The highly conserved asparagine at position 28 in Fig. 2 seems to form a turn at the end of helix 2
of the LysM (Bateman and Bycroft, 2000).
The structures of the three LysMs in the receptor-like
kinase NFP of the legume Medicago trunculata, using
hydrophobic cluster analysis plots, showed that the first
LysM is positively charged or neutral, but that the second
two motifs are predominantly electronegative (Arrighi
et al., 2006). Nodulating plants, like M. trunculata and
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 838–847
LysM, a widely distributed glycan binding domain 841
Table 1. Overview of the number (nr) and the relation between the N- or C-terminal (term) location of LysMs and the specificity of peptidoglycan
hydrolases.
Specificity
Pfam
Locationa
N-term (nr)
C-term (nr)
Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase
N-acetylmuramoyl-L-alanine amidase
Phage lysozyme
Glycosyl hydrolases family 18
Membrane-bound lytic murein transglycosylase D
Transglycosylase SLT domain
Glycosyl hydrolases family 25
Plant self-incompatibility protein S1
NlpC/P60 family
Cysteine, Histidine-dependent Amidohydrolases/Peptidases (CHAP)
Peptidase family M23
Zinc carboxypeptidase
ErfK/YbiS/YcfS/YnhG
Bacterial Ig-like domain (group 1)
Bacterial Ig-like domain (group 2)
PF01832
PF01520
PF00959
PF00704
PF06474
PF01464
PF01183
PF05938
PF00877
PF05257
PF01551
PF00246
PF03734
PF02369
PF02368
Sec
Sec
Sec
Sec
Lipo
Mem
Sec
3
5
1
1
1
2
1
1
1
1
Sec/Lipo
Sec
Sec/Lipo/Mem
Sec
Sec/Lipo/Mem
Mem
Sec/Mem
3
4
1
1
1
1
1
1
1
or 12
till 5
or 2
till 4
or 2
till 3
till 6
till 4, 8 or 9
till
till
till
till
4, or 12
4, 8
8
3
1 or 2
or 2
a. Sec, secreted protein; Mem, membrane protein; Lipo, lipoprotein. In grey the groups containing the highest number of proteins with a certain
specificity are indicated.
Lotus japonicus, are able to recognize specific Nod
factors, which are secreted by their symbiotic bacteria.
Leucine 118 of the second LysM of NFR5 of L. japonicus
is the main determinant in the recognition of the Nod
factor of Rhizobium leguminosarum biovar viciae strain
DZL. This Leu-118 corresponds to residue 5 in Fig. 2. The
DZL Nod factor is not recognized by Lotus filicaulis NFR5,
which has a lysine at position 118 (Radutoiu et al., 2007).
Structural studies should elucidate how Leu-118 determines this specificity.
Mulder et al. (2006) also describe the homology modelling of the three LysMs of M. trunculata using the MltD
LysM structure. They docked chito-oligosacharides and
Nod factors on these models to predict the most
favoured binding modes of these compounds. The Nod
factors specific for M. trunculata are sulphated and
O-acetylated and seem to bind in similar orientation to
all three LysMs. The sulphate group of the Nod factor is
in close proximity to a Lys residue, allowing a salt bridge
between the two. The O-acetyl group of the GlcNAc in
the Nod factor is located in the hydrophobic pocket of
the LysM, made mainly by amino acids of the loop
between strand beta1 and helix alpha1. The lipid moiety
of the Nod factor wraps around the LysM and seems to
interact with amino acids that are at the base of beta2.
The M. trunculata LysMs are highly glycosylated but the
glycosylation does not interfere with Nod factor binding.
Ohnuma et al. (2008) characterized the carbohydrate
binding site of the two LysMs of the chitinase of
P. ryukyuensis using calorimetric and NMR techniques.
They identified residues critical for (GlcNAc)5 binding by
titrating the (GlcNAc)5 and monitoring by NMR. The
N-terminal part of helix 1 and the loop between strand 1
and helix 1, together with the C-terminal part of helix 2
and the loop between helix 2 and strand 2, form a
shallow groove by a cluster of hydrophobic residues.
Moreover, a Tyr residue (at position 10 in Fig. 2) is of
critical importance in (GlcNAc)5 binding.
It will be a challenge to elucidate the structure and
specific interactions of a LysM (protein) bound to its substrate, especially one containing multiple LysMs.
Multiple functions of LysM-containing proteins
Peptidoglycan hydrolases
Most of the LysM-containing proteins are bacterial
PG hydrolases (Fig. 1). Interestingly, LysM domains
of glycosylases, such as N-acetylmuramidases and Nacetylglucosaminidases, are located downstream of the
active-site domain at the C-terminus of the protein. In PG
endopeptidases, such as those of the Cysteine, Histidinedependent Amidohydrolases/Peptidases (CHAP) superfamily (Layec et al., 2008), they are present in the
N-terminal part upstream of the active site (Table 1). A
possible reason for the different topology could be proper
positioning of the active-site domains towards their specific substrates.
The number of LysMs in PG hydrolases and their location in the proteins can vary considerably (Table 1 and
Table S1). The presence of three C-terminal LysMs in the
LysM domain of the N-acetylglucosaminidase AcmA of
L. lactis appears to be optimal for lactococcal cell wall
degradation (Steen et al., 2005a); AcmA derivatives containing one, two or four LysMs bound less efficiently to
L. lactis cells, resulting in reduced cell lysis. When all
LysMs were removed, PG binding was lost, concomitant
with an almost total loss of PG hydrolysing activity.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 838–847
842 G. Buist, A. Steen, J. Kok and O. P. Kuipers 䊏
Similarly, removal of the C-terminal six LysMs from the
orthologous N-acetylglucosaminidase AtlA of E. faecalis
led to a 580-fold reduction of enzyme activity (Eckert
et al., 2006). Whether the number of motifs is optimized
for a specific substrate is unknown.
AcmA is subject to proteolytic degradation by the extracellular proteases PrtP and HtrA (Buist et al., 1998;
Poquet et al., 2000; Steen et al., 2005b). Most likely, the
protein is cleaved within the Ser/Thr/Asp-rich intervening
sequences between the LysMs. The consequent reduction of the number of LysMs results in a reduced enzymatic activity of AcmA. Proteolytic degradation of LysMs
from PG hydrolases is generally observed (Steen et al.,
2005b; Eckert et al., 2006; Fukushima et al., 2006);
whether this degradation within the LysM domain is a
directed manner to control the potentially lethal cell wall
degrading activity is an interesting but as yet unproven
strategy.
Actinobacteria can enter a state in which they are less
metabolically active and lose culturability, a process that
is controlled by resuscitation-promoting factors (RPFs).
Rpf of Micrococcus luteus is essential: it has been
shown to posses muralytic activity that is probably
responsible for its observed activity in resuscitation
(Mukamolova et al., 2006). Rpf belongs to the LysM subfamily of the firmicute Sps proteins, of which all
members contain one C-terminal LysM. Proteins of the
SpsA subfamily contain two N-terminal LysMs (Ravagnani et al., 2005). Proteins of both subfamilies have
been suggested to be secreted, muralytic enzymes that
control bacterial culturability via enzymatic modification
of the bacterial cell envelope.
Phage lysins
LysM domains are also present in bacteriophage lysins
(muramidases) such as those from L. lactis phage Tuc
2009 and the B. subtilis phages phi 29, PBSX and PZA
(Buist et al., 1995). Prophage PBSX encodes the lysins
XkdP (specificity unknown), XlyA and XlyB (both amidases), which each contain one LysM. The LysMs in XlyA
and XlyB are combined with the domain PG_binding_1.
The presence of this additional substrate-binding domain
suggests that each protein binds to two different regions
of the PG substrate to properly position the active-site
domain(s).
Virulence factors
Several LysM domain-containing proteins are virulence
factors of human bacterial pathogens. Staphylococcus
aureus produces five LysM proteins which are all
involved in virulence (Table S1). The mature
N-acetylmuramyl-L-alanine amidase Sle1 of S. aureus
contains three N-terminal LysMs and is involved in cell
separation (Kajimura et al., 2005). Cells of a sle1 mutant
form clusters and the strain is significantly attenuated in
an acute-infection mouse model, indicating that cluster
formation affects spread of the bacteria during infection.
The S. aureus membrane protein EbpS contains an
N-terminal elastin-binding domain for binding to tissue
cells, which is exposed to the cell surface and one
C-terminal LysM, which is not exposed on the cell
surface and was suggested to be buried within the PG
(Fig. 3) (Downer et al., 2002). The covalently PG-bound
protein A of S. aureus also contains a LysM immediately
upstream of the LPxTG box (Bateman and Bycroft,
2000), suggesting an additional binding and/or positioning mode (Fig. 3). Listeria monocytogenes expresses six
LysM proteins (Bierne and Cossart, 2007). The P60 PG
hydrolase contains an N-terminal and a central LysM,
which are separated by an SH3 domain that has a putative PG binding function. Mutations in p60 and namA
(three C-terminal LysM motifs) resulted in intermediate
reduction of bacterial virulence in vivo (Lenz et al., 2003).
The authors speculated that the combined action of the
two PG hydrolases results in the production of glucosaminylmuramyl dipeptide, which is known to modify
host inflammatory responses. The surface immunogenic
protein (Sip) of Streptococcus agalactiae, which is
conserved in strains of group B Streptococcus (GBS),
contains one N-terminal LysM (Borges et al., 2005).
Administration of Sip-specific antibodies to pregnant
mice resulted in protection of the pups against a lethal
challenge with GBS (Martin et al., 2002). Sip was only
protective against GBS strains without a polysaccharide
capsule, showing that accessibility of Sip, which is
exposed on the cell surface at the polar sites and the
septal region (Rioux et al., 2001), can be blocked by
such capsules. The LysM domain-containing proteins
FsaP, TspA and Intimin of, respectively, Francisella
tularensis (Mellilo et al., 2006), Neisseria meningitidis
(Oldfield et al., 2007) and enterohaemorrhagic and
enteropathogenic E. coli (Bateman and Bycroft, 2000)
are involved in adherence to human cells. All three proteins are located in the outer membrane (Fig. 3). The
mature proteins have similar modular structures, in which
a centrally located transmembrane domain is preceded
by a LysM domain, suggesting that the N-terminal part of
the proteins is in contact with the PG in the periplasm
and the C-terminal part is exposed on the cell surface. It
is unknown why these proteins interact with the PG. A
virulent strain of F. tularensis expresses elevated levels
of FsaP and F. tularensis-infected mice developed antibodies against FsaP. Mutation of tspA in N. meningitidis
resulted in reduced binding to Hep-2 and meningothelial
monolayers (Melillo et al., 2006).
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 838–847
LysM, a widely distributed glycan binding domain 843
LysM domain-containing proteins of eukaryotes
LysM domains are also present in (putative) proteins of
plants, fungi and animals, including man. Only some of
the LysM proteins have been studied, e.g. the aforementioned chitinases of C. elegans, but the function of most of
these proteins is still unknown. As mentioned before the
chitinase of C. elegans has several LysMs that contain
consensus Cys residues. The presence of Cys residues
suggests that disulphide bridges are used to maintain
protein structure of the motif (Bateman and Bycroft,
2000), or alternatively could allow for cofactor binding.
The legume L. japonicus has two LysM-type serine/
threonine receptor kinases, NFR1 and NFR5, which
enable the plant to recognize its bacterial symbiont
Mesorhizobium loti (Fig. 3) (Madsen et al., 2003;
Radutoiu et al., 2003). M. trunculata contains seven LysM
domain-containing receptor-like kinases. The authors
speculate that also in this plant species the LysM domains
are involved in recognition of the bacterial symbiont
(Limpens et al., 2003). The symbiotic bacteria secrete
Nod factors, which resemble chitin and are thought to bind
to the LysM domains of the plant receptors. Eventually,
this would lead to endocytosis of the bacteria by plant
cells and subsequent nodulation in the plant roots.
Besides these receptor kinases involved in symbiotic signalling, plant LysM proteins are also involved in defence
signalling against fungal attack. Chitin is involved in inducing defence responses in both monocots and dicots.
Recently it has been shown that CEBiP of Oryza sativa
contains two LysMs and is a glycoprotein with high
affinity for chitin oligosaccharides. Knock-down of
CEBiP resulted in suppression of chitin-induced defence
response (Kaku et al., 2006). Arabidopsis CERK1 is a
receptor-like kinase with three LysMs that also plays a key
role in fungal perception by the plant (Miya et al., 2007).
Localized cellular binding of LysM domains
Although PG covers the entire bacterial cell surface,
immunofluorescence studies showed that the C-terminal
LysM domain of AcmA binds to specific sites on the bacterial surface, i.e. near the poles and septum of L. lactis
cells (Steen et al., 2003). Interestingly, our recent results
show that protein secretion also takes place near the
septum (Buist et al., 2006). The secondary polymers
lipoteichoic acids (LTAs), which consist of polyglycerolphosphate, are responsible for the specific binding of
AcmA: AcmA does not bind where LTAs are present
(Steen et al., 2003). Also the presence of surface layer
proteins hinders the binding of the LysM domain of AcmA
to Lactobacillus helveticus (Steen et al., 2003) and, thus,
localization of binding was found to be species-specific.
Increased O-acetylation of the PG of L. lactis results in
resistance of the cells to AcmA, possibly by reduced
binding of AcmA (Veiga et al., 2007), while de-Nacetylation of lactococcal PG did not affect the binding
of AcmA (Meyrand et al., 2007). Interestingly, Aaa of
S. aureus, which has three N-terminal LysMs, seems to
bind all over the cell surface (Heilmann et al., 2005).
The LysM domain of Sip of S. agalactiae is responsible
for localized binding of the protein at the cell poles and the
septal region (Rioux et al., 2001). Polar and septal binding
has also been shown for the D,L-endopeptidases LytE,
CwlS and LytF of the rod-shaped bacterium B. subtilis.
These proteins contain three, four and five N-terminal
LysMs respectively (Yamamoto et al., 2003; Fukushima
et al., 2006). LytE, prior to secretion, interacts with
membrane-associated MreBH, which is required for the
helical pattern of extracellular localization of LytE into the
cylindric part of the cell wall (Carballido-Lopez et al.,
2006). The pattern of LytE localization resembles that of
the SecA and SecY proteins of the general Sec-secretion
machinery of B. subtilis (Campo et al., 2004). No interaction of MreBH with the latter two proteins was found.
Whether localization of secretion of the LysM proteins
coincides with their location of activity is also something
that needs further study.
The PG hydrolase YneA of B. subtilis is responsible for
suppression of cell division during SOS response. YneA
consists of only 105 aa and has a putative N-terminal
transmembrane domain that is separated by only 10 aa
from a centrally located LysM (Kawai et al., 2003). This
suggests that YneA is inserted into the cytoplasmic membrane and binds to the lower layers of the PG (Fig. 3). The
pH in the PG matrix of respiring cells of B. subtilis is lower
than that of the cell surface (Calamita and Doyle, 2002).
Taking these observations together it can be postulated
that binding of the LysM of YneA is solely possible in the
PG matrix and this is possibly a general principle for
LysMs with a low pI. Recently, it has been shown that the
cell walls of Gram-positive bacteria consist of two zones:
the so-called inner-wall and outer-wall zone, of which the
first resembles the periplasmic space of Gram-negative
bacteria (Matias and Beveridge, 2005). Whether extracellular or membrane-associated proteins with LysM
domains of Gram-positive bacteria with a pI below 7
reside in the inner wall zone and those with a pI above 7
bind to the outer cell surface is an interesting possibility.
Why certain membrane proteins and proteins that are
covalently bound to the cell wall have an additional LysM
for non-covalent PG binding is currently unknown. It could
be that proper positioning of active-site domains(s) at the
desired location in or on the cell wall requires the LysM
domain(s) as the retention signal.
The membrane protein Rv2719c of Mycobacterium
tuberculosis contains a short transmembrane segment
and a C-terminal LysM and shows homology to YneA of
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 838–847
844 G. Buist, A. Steen, J. Kok and O. P. Kuipers 䊏
B. subtilis (Chauhan et al., 2006). Rv2719c has cell
wall hydrolase activity, is a potential regulator of
M. tuberculosis cell division and the amount of the protein
and, possibly, its activity are modulated under a variety of
growth conditions. Localization of GFP–Rv2719c fusion
protein led to the suggestion that Rv2719c is targeted to
potential PG synthesis zones, i.e. to the poles and to sites
close to mid-cell.
LysM domains also play a role in the development of
spores in sporulating bacteria. When the signal peptide
of E. coli b-lactamase was replaced by the two LysMs of
the B. subtilis spore protein YaaH, the fusion protein was
directed to the surface of the developing spore. Apparently, the LysM domains of spore proteins serve as localization signals to forespores. SafA and SpoVID localize
to the cortex–coat interface encircles the entire cortex
layer of the spore (Fig. 3) (Costa et al., 2006). SafA is
initially targeted independently of SpoVID, possibly via its
LysM domain, while in a second stage SafA forms a
complex with SpoVID via direct protein–protein interactions. Both proteins promote attachment of the spore
coat to the spore cortex and both proteins and their
complex are likely to interact with additional coat components (Costa et al., 2006). Interestingly SafA and
SpoVID have a rather low pI; whether this is important
for localized binding in the spore coat remains to be
answered. Localized and controlled binding of spore proteins to the cortex–coat interface via LysM domains, as a
general concept to induce protein complex formation in
the spore coat, is a very interesting model but remains to
be further investigated.
Together these findings show that proteins containing a
LysM domain bind PG at specific loci depending on the
presence of secondary cell wall polymers and, possibly,
PG modifications.
Evolution of LysM domains
The presence of LysMs in many bacterial species as well
as many eukaryotes, and the absence of the motif in
archaeal proteins raises questions about their origin and
evolution. Two theories can be envisaged: LysM was
present in the common ancestor of all life, but was lost in
the archael lineage, or it evolved after the separation of
bacteria, archaea and eukaryotes and was subsequently
transferred from bacteria to eukaryotes, or vice versa
(Ponting et al., 1999). Because of their omnipresence in
the latter kingdoms, the second theory requires that the
transfer occurred early in evolution, or later via multiple
transfers. Phylogenetic information is, however, not sufficient to resolve the earliest relationships between LysM in
bacteria and eukaryotes. In bacteria, the LysM seems to
have evolved into a general PG-binding motif, whereas in
eukaryotes it is a chitin-binding motif.
Motif similarities suggest that plant LysMs are ancient
and have evolved through local and segmental
duplications. The family has undergone further duplication
and diversification in legumes, where some LysM kinases
function as receptors for bacterial nodulation factors.
Plant LysMs eventually evolved into a minimum of six
distinct types in LysM kinases and five additional types in
non-kinase proteins (Zhang et al., 2007).
Alignment of bacterial LysM domains with two other
carbohydrate-binding motifs in proteins revealed a shared
YG motif (Turner et al., 2004). The cholin-binding
domains of cell surface proteins binding to cholin in LTA
of Streptococcus pneumoniae and Clostridia species, as
well as the LTA-binding GW domain of LTA-binding proteins of Listeria species, also contain the YG motif (Fig. 2).
It is possible that an ancestral carbohydrate-binding
domain in Gram-positive bacteria has evolved into LysM,
choline binding and GW domains. Sugar-binding proteins
usually have aromatic residues, which enable contact
between the protein surface and the sugar. The YG motif
could therefore also have been the result of parallel
evolution.
Concluding remarks
The LysM domain is a widely spread domain in nature that
has been shown to bind different PG types in bacteria,
chitin-like compounds in eukaryotes and a viral
glycoprotein. GlcNAc seems to be an important constituent of the LysM ligand and has been shown to interact
with LysMs of some eukaryotic proteins. Whether GlcNAc
is the sole moiety recognized by LysM remains to be
elucidated. In bacterial PG hydrolases, the motif
is possibly needed to properly position the active-site
domain(s) towards their substrate (Steen et al., 2005a). In
legume plants, LysM plays a crucial role in establishing
the interaction of the plant with nitrogen-fixing bacteria by
binding Nod factors secreted by the bacteria. In other
plants, a defence response against fungi is induced
via binding of GlcNAc to LysM-containing receptor-like
kinases. For other LysM-containing proteins the nature of
ligand still has to be established, for both the entire LysM
protein and its individual LysM(s). Especially, the LysM
domains of animal and fungal proteins need attention.
The structure of only two LysMs is known and more
structures need to be determined. Until then, modelling of
LysM structures is helpful to establish how and where the
ligand binds. Eventually, crystal or NMR structures of
prokaryotic and eukaryotic LysMs together with their specific ligands will be essential to fully understand ligand
binding.
LysM is often found in multiple copies in proteins and
the exact function of these duplications also needs attention, as only for the L. lactis autolysin AcmA has it been
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 838–847
LysM, a widely distributed glycan binding domain 845
shown that three LysMs are optimal for proper enzyme
functioning. Characteristics of the LysM domains such as
their pI, glycosylation, presence of disulfide bridges, aa
insertions compared with the consensus sequence
(Fig. 2) and composition and length of the intervening
sequences between the LysMs need to be investigated.
More is known about the subcellular localization of
LysM proteins in bacteria, an important aspect as, e.g. in
the case of AcmA, this process seems to regulate the
potentially lethal activity of the autolysin. The subcellular
localization of the proteins is directed by their LysM
domains and further influenced by cell wall components
such as other proteins, LTA and modifications of the PG.
Future research should address the (combined) effects of
these cell wall components on localization at the molecular level.
Finally, as most of the LysM proteins are secreted and
bind PG, it will be interesting to establish the impact of
possible intercellular action, such as binding of the proteins
to other cells, for instance, for in trans cell lysis between
species in mixed cultures, aggregation and biofilm formation. An example of biotechnological application of LysMs
is the use of the LysM domain of AcmA to bind antigens to
non-genetically modified Gram-positive bacteria for oral
immunization purposes (Bosma et al., 2006; van Roosmalen et al., 2006). To that end, the LysM domain was
fused to the C-terminus of antigens of the malaria parasite
Plasmodium falciparum or S. pneumoniae, and produced
and secreted by L. lactis. The secreted antigen–LysM
fusion proteins could subsequently bind to non-genetically
modified bacteria or their purified cell walls. After oral or
subcutaneous immunizations of mice with the immobilized
recombinant antigens, a specific, possibly protective, antibody response was obtained. Many more applications, e.g.
in modulating host–microbe interactions and surface
display of enzymes based on the specific binding properties of the LysMs, are foreseen.
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