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Research Article
Structures of SALSA/DMBT1 SRCR domains reveal
the conserved ligand-binding mechanism of the ancient
SRCR fold
Martin P Reichhardt
1
, Vuokko Loimaranta
2
, Susan M Lea
1,3
, Steven Johnson
1
The scavenger receptor cysteine-rich (SRCR) family of proteins
comprises more than 20 membrane-associated and secreted
molecules. Characterised by the presence of one or more copies of
the ~110 amino-acid SRCR domain, this class of proteins have
widespread functions as antimicrobial molecules, scavenger re-
ceptors, and signalling receptors. Despite the high level of struc-
tural conservation of SRCR domains, no unifying mechanism for
ligand interaction has been described. The SRCR protein SALSA, also
known as DMBT1/gp340, is a key player in mucosal immunology.
Based on detailed structural data of SALSA SRCR domains 1 and 8,
we here reveal a novel universal ligand-binding mechanism for
SALSA ligands. The binding interface incorporates a dual cation-
binding site, which is highly conserved across the SRCR superfamily.
Along with the well-described cation dependency on most SRCR
domain–ligand interactions, our data suggest that the binding
mechanism described for the SALSA SRCR domains is applicable to
all SRCR domains. We thus propose to have identified in SALSA a
conserved functional mechanism for the SRCR class of proteins.
DOI 10.26508/lsa.201900502 | Received 26 July 2019 | Revised 14 February
2020 | Accepted 14 February 2020 | Published online 25 February 2020
Introduction
The salivary scavenger and agglutinin (SALSA), also known as gp340,
“deleted in malignant brain tumors 1”(DMBT1) and salivary ag-
glutinin (SAG), is a multifunctional molecule found in high abun-
dance on human mucosal surfaces (1,2,3,4). SALSA has widespread
functions in innate immunity, inflammation, epithelial homeosta-
sis, and tumour suppression (5,6,7). SALSA binds and agglutinates a
broad spectrum of pathogens including, but not limited to, human
immunodeficiency virus type 1, Helicobacter pylori,Salmonella
enterica serovar Typhimurium, and many types of streptococci (8,9,
10,11). In addition to its microbial scavenging function, SALSA has
been suggested to interact with a wide array of endogenous im-
mune defence molecules. These include secretory IgA, surfactant
proteins A (SP-A) and D (SP-D), lactoferrin, mucin-5B, and com-
ponents of the complement system (1,2,12,13,14,15,16,17,18).
SALSA thus engages innate immune defence molecules and has
been suggested to cooperatively mediate microbial clearance and
maintenance of the integrity of the mucosal barrier.
The 300- to 400-kD SALSA glycoprotein is encoded by the DMBT1
gene. The canonical form of the gene encodes 13 highly conserved
scavenger receptor cysteine-rich (SRCR) domains, followed by two
C1r/C1s, urchin embryonic growth factor and bone morphogenetic
protein-1 (CUB) domains that surround a 14
th
SRCR domain, and
finally a zona pellucida domain at the C terminus (19,20). The first 13
SRCRs are 109 aa domains found as “pearls on a string”separated
by SRCR-interspersed domains (SIDs) (Fig 1A)(1,21). The SIDs are 20-
to 23-aa-long stretches of predicted disorder containing a number
of glycosylation sites, which have been proposed to force them into
an extended conformation of roughly 7 nm (7). In addition to this
main form, alternative splicing and copy number variation mech-
anisms lead to expression of variants of SALSA containing variable
numbers of SRCR domains in the N-terminal region.
The SRCR protein superfamily include a range of secreted and
membrane-associated molecules, all containing one or more SRCR
domains. For a number of these molecules, the SRCR domains have
been directly implicated in ligand binding. These include CD6 sig-
nalling via CD166, CD163-mediated clearance of the haemoglobin–
haptoglobin complex, Mac-2 binding protein’s (M2bp’s) interaction
with matrix components, and the binding ofmicrobial ligands by the
scavenger receptors SR-A1, SPα, and MARCO (22,23,24,25,26,27).
Although the multiple SALSA SRCR domains likewise have been im-
plicated in ligand binding, the molecular basis for its diverse inter-
actions remains unknown.
To understand the multiple ligand-binding properties of the
SALSA molecule, we undertook an X-ray crystallographic study to
provide detailed information of the SALSA interaction surfaces. We
here provide the atomic resolution structures of SALSA SRCR do-
mains 1 and 8. We identify cation-binding sites and demonstrate
their importance for ligand binding. By comparing our data to
previously published structures of SRCR domains, we propose a
1
Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
2
Institute of Dentistry, University of Turku, Turku, Finland
3
Central Oxford Structural Molecular
Imaging Centre, University of Oxford, Oxford, UK
Correspondence: steven.johnson@path.ox.ac.uk; martinpreichhardt@gmail.com
©2020Reichhardt et al. https://doi.org/10.26508/lsa.201900502 vol 3 | no 4 | e201900502 1of10
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generalised binding mechanism for this ancient, evolutionarily
conserved, fold.
Results
The scavenger receptor SALSA has a very wide range of described
ligands, including microbial, host innate immune, and ECM mole-
cules. To understand the very broad ligand-binding abilities of the
SALSA molecule, we applied a crystallographic approach to deter-
mine the structure of the ligand-binding SRCR domains of SALSA.
SRCR domains 1 and 8 (SRCR1 and SRCR8) were expressed in Dro-
sophila melanogaster Schneider S2 cells with a C-terminal His-tag.
The domains were purified by Ni-chelate and size-exclusion chro-
matography (SEC) and crystallised, and the structures were solved by
molecular replacement. This yielded the structures of SRCR1 and
SRCR8 at 1.77 and 1.29
˚
A, respectively (Fig 1B). (For crystallographic
details, see Table 1).
The SALSA SRCRdomains reveal a classic globular SRCR-fold, with
four conserved disulphide bridges, as described for the SRCR type B
domains. The fold contains one α-helix and one additional single
helical turn. The N and C termini come together in a four-stranded
β-sheet. SALSA SRCR1-13 are highly conserved, with 88–100% identity.
Variation is only observed in 9 of the 109 aa residues, all of these
observed in peripheral loops, without apparent structural signifi-
cance. Combined, the data from SRCR1 and SRCR8 are thus valid
representations of all SALSA SRCR domains. Both SRCR1 and SRCR8
are stabilized by a metal ion buried in the globular fold. The
placement suggests the ion is bound during the folding of the do-
main and is modelled as Mg
2+
, which is present in the original ex-
pression medium and in the crystallisation conditions of SRCR1.
So far, all described ligand-binding interactions of SALSA have
been shown to be Ca
2+
-dependent. We therefore proceeded to ad-
dress the ligand-binding potential of the SRCR domains by adding
Ca
2+
,Mg
2+
, and a cocktail of sugars to SRCR8 crystals before freezing.
This yielded a second crystal form ofSRCR8 with the original Mg
2+
ion,
site 1, and two additional cations bound, sites 2 and 3 (Fig 2). All three
sites are class three cation-binding sites, with the coordination
obtained from residues in distant parts of the sequence (28).
Assignment of the identity of the ions at the paired site was carried
out by modelling Mg
2+
or Ca
2+
at each site, followed by refinement of
the structure and analysis of the difference maps (Fig S1). These
revealed that Mg
2+
best satisfied the data at both sites, consistent with
the 20-fold molar excess of Mg
2+
over Ca
2+
in the crystallisation so-
lution. However, it is worth noting that either site could likely ac-
commodate either cation depending on local concentration. Analysis
of the bond lengths and coordination numbers suggest that site 2 is a
canonical Mg
2+
site, with octahedral geometry and average bond
lengths of 2.1
˚
A,whilesite3displaysahighercoordinationnumberand
longer bond lengths, more consistent with a Ca
2+
-binding site (29). The
Mg
2+
at site 1 is coordinated by the backbone carbonyl groups of S1021
and V1060, as well as the side chains of D1023 and D1026, and two
waters, and is buried in the domain fold (Fig 2C). The Mg
2+
at site 2 is
coordinated by D1019, D1020, and E1086 plus three waters (Fig 2D). The
Mg
2+
at site 3 is coordinated by the side chains of D1020, D1058, D1059,
and N1081, with additional contributions from a water and an extra
density (Fig 2E). Attempts to model this extra density as any of the
sugars or alcohols present in the crystallisation solution failed to
produce a satisfactory fit; therefore, it likely represents a superposition
of a number of molecules.
In contrast to the Mg
2+
at site 1, these cations at sites 2 and 3 are
exposed on the surface of the domain, and the protein only con-
tributes a fraction of the coordination sphere, with the remainder
contributed by waters or small molecules from the crystallisation
solution. According to the literature, the majority of described SALSA
ligands are negatively charged. Thus, the surface-exposed cations
likely provide a mechanism for ligand binding for the SALSA SRCR
domains, whereby the anions of the ligand substitute for the waters
or the density at site 3 observed in our structure. To test this hy-
pothesis, site-directed mutagenesis was used, targeting the key
residues coordinating sites 2 and 3. Included in the further analysis
were single mutations D1019A and D1020A. While mutation of D1019 is
expected to only disrupt binding of cations at site 2, mutation of the
shared D1020, will likely affect binding of both cations.
As SALSA recognizes a very broad range of biological ligands, we
set out to test the effect of SRCR domain point mutations on in-
teractions with a wide array of biological ligands. These included
binding to (1) hydroxyapatite, a phosphate-rich mineral essential for
the binding of SALSA to the teeth surface, where it mediates anti-
microbial effects (30); (2) heparin, a sulphated glycosaminoglycan as
a mimic for theECM/cell surface, forwhich binding of SALSA hasbeen
described to affect cellular differentiation and microbial colonisation
(31); (3) Group A Streptococcus surface protein, Spy0843, a leucine-
rich repeat protein demonstrated to bind to SALSA (32)(Fig 3).
Figure 1. Crystal structure of SALSA domains SRCR1 and SRCR8.
(A) Schematic representation of the domain organization of full-length SALSA.
SRCR1 and SRCR8 are highlighted in green and blue, respectively. All SRCR
domains share >88% sequence identity. 100% identity is shared by SRCR3 and 7
(yellow) and SRCR10 and 11 (purple). (B) Front and side views of an overlay of
SRCR1 (green) and SRCR8 (blue), showing four conserved disulphide bridges
(yellow). Both SRCR1 and SRCR8 were found to coordinate a metal ion, modelled as
Mg
2+
(dark green for SRCR1 and dark blue for SRCR8). The limited structural
variation observed between SRCR1 and SRCR8 (92% sequence identity) imply that
these are appropriate representations of all SALSA SRCR domains 1–13.
SALSA domain structures Reichhardt et al. https://doi.org/10.26508/lsa.201900502 vol 3 | no 4 | e201900502 2of10
Different binding assays provide an understanding of a gener-
alised binding mechanism of SALSA SRCR domains. While the WT
SRCR domain bound to all three ligands, both of the cation-binding
site mutations, D1019A and D1020A, abolished binding. This is
consistent with the bound cations acting as a bridge for ligand
interaction and thus provides a mechanistic explanation for the
binding properties of the SALSA SRCR domains. In the literature,
SALSA ligand binding has been described as specifically calcium
dependent. To verify this, we conducted binding assays in an
MgEGTA-containing buffer (Fig 3C). The exchange of magnesium for
calcium abolished ligand interactions, thus supporting a calcium-
specific mediation of binding. As mutation of site 2 alone (modelled
as Mg
2+
in our structure) abolished binding, our data suggest that
Ca
2+
may occupy site 2 under physiological conditions.
All known members of the SRCR superfamily share a very high
degree of identity, both at the sequence and structural levels. An
FFAS search (33) of the SRCR8 sequence showed highest similarities
to CD163 SRCR5 (score: −65.4, 46% identity), M2bp (score: −64.4, 54%
identity), neurotrypsin (score: −61.5, 50% identity), MARCO (score:
−59.4, 50% identity), CD5 SRCR1 (score: −48.8, 26% identity), and CD6
SRCR2 (score: −45.6, 60% identity). Using the Dali server (34), searches
for the cation-binding SRCR8 soak structureidentified two top hits as
M2bp (pdbid: 1by2) and CD6 SRCR3 (pdbid: 5a2e). These were iden-
tified with respective Z-scores of 21.4 (r.m.s.d. of 1.1
˚
Awith106of112
residues aligned) and 20.4 (r.m.s.d. of 1.5
˚
A with 109 of 109 residues
aligned). Despite the classical division of SRCR superfamily proteins into
groups A and B, based on the conserved three versus four cysteine
bridges,theSRCRfoldisveryhighlyconserved,andtheSALSASRCR
domain structures correlate closely to both group A and group B SRCR
superfamily domains (Fig 4A).
For members of the SRCR superfamily where the SRCR domain
directly partakes in ligand binding, both microbial and endogenous
protein ligands have been described. For MARCO, crystallographic
structures identified a cation-binding site exactly corresponding to
Table 1. Data collection and refinement statistics (molecular replacement).
SRCR1 (pdbid: 6sa4) SRCR8 (pdbid: 6sa5) SRCR8soak (pdbid: 6san)
Data collection
Space group P 2
1
2
1
2
1
P2
1
2
1
2
1
P12
1
1
Cell dimensions
a,b,c(
˚
A) 36.77, 45.19, 69.37 32.82, 40.82, 62.99 27.24, 46.64, 93.63
α,β,γ(°) 90.00, 90.00, 90.00 90.00, 90.00, 90.00 90.00, 97.37, 90.00
Resolution (
˚
A) 28.52–1.77 (1.80–1.77)
a
40.82–1.29 (1.31–1.29) 46.64–1.36 (1.39–1.36)
R
merge
0.17 (1.36) 0.117 (1.12) 0.074 (0.801)
I/σI8.1 (1.1) 8.6 (0.9) 14.9 (2.2)
Completeness (%) 99.8 (99.3) 100 (99.6) 98.3 (96.9)
Redundancy 6.3 (6.6) 11.4 (8.0) 6.6 (6.4)
Refinement
Resolution (
˚
A) 28.52–1.77 (1.95–1.77) 34.27–1.29 (1.35–1.29) 30.95–1.36 (1.39–1.36)
No. of reflections 11,730 21,958 49,166
R
work
/R
free
0.186/0.229 (0.266/0.348) 0.155/0.188 (0.326/0.279) 0.186/0.226 (0.267/0.326)
No. of atoms
Protein 824 829 3,192
Ligand/ion 26 18 34
Water 109 124 358
B-factors
Protein 22.36 16.80 17.48
Ligand/ion 49.33 54.03 26.08
Water 31.15 33.66 35.55
R.m.s. deviations
Bond lengths (
˚
A) 0.006 0.009 0.007
Bond angles (°) 0.809 1.026 0.87
Ramachandran outliers 0 0 0
Rotamer outliers 0 0 0
Number of crystals was one for each structure.
a
Values in parentheses are for highest resolution shell. Data from SRCR1 and SRCR8 crystals were collected on Diamond beamline I04, while data for SRCR8soak
were collected on beamline I03.
SALSA domain structures Reichhardt et al. https://doi.org/10.26508/lsa.201900502 vol 3 | no 4 | e201900502 3of10
site 2 in the SALSA SRCR8 domain (26) and point mutations of this site
abolished function. Common to the MARCO and SALSA SRCR domains
is the cluster of negatively charged residues coordinating the
functionally important cations. A similar cluster is also observed in
the SRCR3 domain of CD6 and has been shown by mutagenesis to be
directly involved in binding to the human surface receptor CD166.
Indeed, a point mutation of D291A (corresponding to D1019 of SALSA-
SRCR8) reduced the ligand-binding potential of CD6 to less than 10%
(27). Furthermore, mutational studies of SRCR domains 2 and 3 from
CD163 proved an involvement of this specific site in the binding of the
haemoglobin–haptoglobin complex (36). All structural evidencefrom
mutational studies of SRCR domains thus indicate a conserved
surface-mediating ligand binding (Fig 4B).
Interestingly, various levels of calcium-dependency on ligand
interactions have been described for all SRCR domains directly
involved with binding. SR-A1, Spα,MARCO,CD5,andCD6allrelyon
calcium for interactions with microbial ligands (24,25,26,37,38,39).
Furthermore, the binding of CD163 to the haemoglobin–haptoglobin
complex is calcium-dependent, while CD6 also recognizes endoge-
nous surface structures (other than CD166) in a calcium-dependent
manner (40). This suggests that the cation-dependent binding
mechanism identified for SALSA is a general conserved feature of all
SRCR domains. Indeed, sequence alignment of SRCR domains from 10
different SRCR superfamily proteins, all with SRCR domains directly
involved with ligand binding, reveals a very high level of conservation
of the two cation-binding sites identified in the SALSA domains (Fig
4C). The ConSurf server is a tool to estimate (on a scale from 1 to 9) the
level of evolutionary conservation of residues in a given fold (41). A
search with the SRCR8 model shows that D1019, D1020, D1058, and
E1086 all score 7 (highly conserved), while N1081 and D1059 score 6
and 4, respectively (thus less conserved). Whenever sequence identity
is not conserved, substitutions are observed with other residues
overrepresented in cation-binding sites (D, E, Q, and N) (28,29). The
cation-binding sites identified in the SALSA SRCR domains, thus,
appear to be a highly conserved feature of the general SRCR fold.
Figure 2. Crystal structure of SRCR8 with bound magnesium ions indicates
mechanism of ligand binding.
(A) Surface charge distribution of SRCR8 (calculated without the presence of
cations) shows a positive cluster on one side with a strong negative cluster
on the other. The negative cluster expands across ~300
˚
A
2
and mediates
the binding of three cations (green). (B) Representation of the residues
coordinating the three cations. The upper Mg
2+
, site 1, sits somewhat buried
in the structure and may be essential for structural stability. The lower
cations at sites 2 and 3 are more exposed. (C) Detailed view of the
coordination of the upper Mg
2+
, site 1. The coordination number of six is
achieved by two waters, two backbone carbonyls, and two side chain
carboxylates. (D) Detailed view of the coordination of the cation at site 2,
modelled as Mg
2+
based on bond length and coordination number. Here, the
coordination number of six is achieved by three waters and three side
chain carboxylates. (E) Detailed view of the coordination of the cation at site
3, modelled as Mg
2+
. The coordination is achieved by three side chain
carboxylates, one side chain amide, and an unmodelled density that is
assumed to be a superposition of crystallisation condition compounds.
Figure 3. Mutating the cation-binding residues of scavenger receptor cysteine-
rich (SRCR) domains abolish function.
Through multiple ligand-binding assays, we demonstrated the functional
importance of cation binding by the SRCR domains. Mutations affecting site 2
(D1019A) and mutations affecting sites 2 and 3 (D1020A) both abolish function.
(A) WT and mutant forms of SRCR8 were incubated with hydroxyapatite beads in a
Ca
2+
-containing buffer. After extensive washing, bound protein was eluted with
EDTA. Eluted fractions were run on a 4–20% SDS–PAGE gel and visualized by
Coomassie staining. Only WT SRCR8 bound hydroxyapatite. (B) WT and mutant
forms of SRCR8 were flown over a heparin (HiTrap HP, 1 ml) column in a Ca
2+
-
containing buffer. Protein bound to the column was eluted with 0.5 M EDTA.
Only WT SRCR8 bound the heparin column. Traces: SRCR8 (blue), D1020A (pink),
D1019A (red), conductivity (brown). (C) In an ELISA-based setup, a concentration
range of the Spy-2 domain of Spy0843 was coated (1–100 μg/ml). WT and
mutant SRCR8 domains were added (100 μg/ml), and binding was detected with a
monoclonal anti-SALSA antibody. Binding was only observed for WT SRCR8. (D)
Overview of ligand-binding studies; + denotes binding, −denotes no binding.
SALSA domain structures Reichhardt et al. https://doi.org/10.26508/lsa.201900502 vol 3 | no 4 | e201900502 4of10
Figure 4. Conserved ligand-binding motif across scavenger receptor cysteine-rich (SRCR) domains.
SRCR domains from multiple proteins engage in cation-depende nt ligand interactions. (A) Structural overlay of domains from seven SRCR superfamily proteins, all with
ligand binding mediated through the SRCR domain. This reveals a highly conserved fold across both type A and type B SRCR domains. SRCR1 (pale green), SRCR8 (light
blue), MARCO (pdbid: 2oy3, sand), CD163 (pdbid: 5jfb, purple), CD5 (pdbid: 2OTT, grey), CD6 (pdbid: 5a2e, pink), M2bp (pdbid: 1by2, yellow), and murine neurotrypsin (pdbid:
6h8m, teal) (35). SALSA magnesium: green, MARCO magnesium: blue. (B) Surface representation of CD6 SRCR3, SALSA SRCR8, and MARCO in same orientation. Point
mutations with a verified impact on ligand binding are highlighted in red, indicating a conserved surface involved in ligand binding. Bound magnesium is highlighted in
SALSA domain structures Reichhardt et al. https://doi.org/10.26508/lsa.201900502 vol 3 | no 4 | e201900502 5of10
Structural inspection of the overlay of SALSA SRCR8 with the corre-
sponding region in the other known SRCR domain structures clearly
shows the potential for cation binding at these sites. We thus propose
that the cation-binding sites identified here are an essential feature of
the ancient SRCR fold and are a conserved mechanism responsible for
mediating ligand binding in the class of SRCR superfamily proteins.
Discussion
Although SALSA has previously been described to interact with a wide
range of biological ligands, little has been known of the binding
mechanisms. Furthermore, it has not been known if SALSA interacts with
various ligands in a similar way or if distinct binding sites are used. Here,
we demonstrate that mutations of a dual cation-binding site interrupt
interactions with a representative selection of very different types of
ligands. Specific disruption of site 2 was sufficient to abolish ligand
binding. We modelled the cation at site 2 in our crystal as Mg
2+
, based on
an analysis of bond length, coordination number, and behaviour of
crystallographic refinements with different cations modelled. However,
experimental data demonstrated that binding to the ligands tested was
only dependent on the presence of Ca
2+
and not Mg
2+
.Thisisinlinewith
previous descriptions of most SALSA–ligand interactions (5,6,42). In the
extracellular compartment, the molar concentration of Ca
2+
is higher
than Mg
2+
(2.5 and 1 mM for calcium and magnesium, respectively), and
it is therefore likely that both sites 2 and 3 will be occupied by Ca
2+
in a
physiological setting. The identification of this dual Ca
2+
-binding site
thus provides an explanation for the Ca
2+
dependency of all SALSA–
ligand interactions described in the literature, suggesting this mech-
anism of binding is applicable to all SALSA SRCR ligands. Multiple
studies have proposed a role for the motif GRVEVxxxxxW in ligand
binding (43,44,45). The crystal data show that this peptide sequence is
buriedintheSRCRfold,andwefoundnovalidationforaroleinligand
binding, although mutations within this sequence are likely to perturb
the overall fold. This motif thus does not appear to have any physi-
ological relevance as defining a ligand-binding site.
The conserved usage of a single ligand-binding area for multiple
interactions suggests that each SALSA SRCR domain engages in one li-
gand interaction. A common feature of the ligands described here, as well
as a number of other ligands such as DNA and LPS, is the presence of
repetitive negatively charged motifs (31). We analysed ligand binding by
individual SRCR domains in surface-plasmon resonance and isothermal
calorimetry assays, but interactions were observed to be of very low
affinity, making reliable measurementsunfeasible.Thisisnotsurprising
for a molecule such as SALSA, where the molecular makeup with the full
extension of 13 repeated units, interspersed by predicted nonstructured
flexible SIDs, provides a molecule that can generate high-avidity inter-
actions with repetitive ligands, despite having only low-affinity interac-
tions for an individual domain. Furthermore, it has been suggested that
SALSA in body secretions may oligomerize into larger complexes (5,46,47,
48), probably via the C-terminal CUB and zona pellucida domains. The
repetitive nature and possible oligomerization allow SALSA to not only
engage with a repetitive ligand on one surface (e.g., LPS or Spy0843 on
microbes) but also engage in multiple ligand interactions simultaneously.
This would be relevant for its interactions with other endogenous de-
fence molecules, such as IgA, SPs, and complement components, where a
cooperative effect on microbial clearance has been demonstrated (12,16,
17,49). In addition, this model of multiple ligand binding would be rel-
evant for microbes described to use SALSA for colonisation of the teeth or
the host epithelium (10,50,51)(Fig 5).
SALSA belongs to the SRCR superfamily, a family of proteins char-
acterised by the presence of one or more copies of the ancient and
evolutionarily highly conserved SRCR fold (52).Althoughacoupleof
SRCR domains, such as the ones found in complement factor I and
hepsin, have not been described to bind ligands directly, most others
have (53,54). SRCR superfamily members, such as SALSA, SR-A1, Spα,
SSc5D, MARCO, CD6, and CD163, have broad scavenger-receptor func-
tions, recognizing a broad range of microbial surface structures and
mediate clearance (24,25,26,37). Although this potentially is relevant for
all SRCR superfamily proteins, some members of the family have
distinctproteinligands,suchasCD6,CD163,andM2bp(22,23,27,55).
With the exception of the CD6–CD166 interactions, most described
SRCR–ligand interactions are calcium dependent, irrespective of the
ligand (24,25,26,37,38,39,40). A cation-binding site is conserved across
SRCR domains, and multiple studies support a role for this site in ligand
binding. Even the specialised CD6–CD166 interaction uses the same
surface for binding, despite “having lost”the calcium dependency (27).
Our studies have thus identified a dual cation-binding site as es-
sential for SALSA–ligand interactions. Analysis of SRCR folds from
various ligand-binding domains reveals a very high level of conser-
vationoftheresiduesatthisdualsite. The conservation of this site,
along with the well-described cation dependency on most SRCR–ligand
interactions, suggests that the binding mechanism described for the
SALSA SRCR domains is applicable to all SRCR domains. We thus
propose to have identified in SALSA a conserved functional mechanism
for the SRCR class of proteins. This notion is further supported by the
specific lack of conservation of these residues observed in the SRCR
domains of complement factor I and hepsin, where no ligand binding
has been shown. The SRCR domains in these two molecules may thus
represent an evolutionary diversion from the common broad ligand-
binding potential of the SRCR fold. The novel understanding of the SRCR
domain generated here will allow for an interesting future targeting of
other SRCR superfamily proteins, with the potential of modifying function.
Materials and Methods
Expression of recombinant proteins
Insect cell expression
Codon-optimized DNA (GeneArt; Thermo Fisher Scientific) was cloned
into a modified pExpreS2-2 vector (ExpreS2ion Biotechnologies) with a
green. (C) Clustal Omega (EMBL-EBI) sequence alignment of SRCR domains from 10 SRCR superfamily proteins. Conservation of the cation-binding sites are displayedingreen
(site 2) and purple (site 3). Dark colouring indicates 100% identity with the SALSA sites, and lighter colouring indicates conservation of residues commonly implicated in cation-
binding (D, E, Q, or N). Cysteines are highlightedin yellow, and over all sequence identity is denoted by *(100%), :(strongly similar chemical properties), and .(weakly similar chemical
properties).
SALSA domain structures Reichhardt et al. https://doi.org/10.26508/lsa.201900502 vol 3 | no 4 | e201900502 6of10
C-terminal His-6 tag. The purified plasmid was transformed into S2
cells grown in EX-CELL 420 (Sigma-Aldrich) with 25 μl ExpreS2 Insect-TR
5X (ExpreS2ion Biotechnologies). Selection for stable cell lines (4 mg/
ml geneticin [Thermo Fisher Scientific]) and expansion were carried
out according to the manufacturer’s instructions.
Escherichia coli expression
DNA strings (GeneArt; Thermo Fisher Scientific) were cloned into
pETM-14 and transformed into M15pRep cells. Protein expression was
carried out in LB media (with 30 μg/ml kanamycin). Cells were in-
duced with 1 mM IPTG. The cultures were centrifuged (3,220g,15min)
and the cell pellets resuspended and lysed in PBS containing 1 mg/
ml DNase and 1 mg/ml lysozyme.
Protein purification
SRCR domains
Insect culture supernatant was collected by centrifugation (1,000gat
30 min), filtered and loaded onto a Roche cOmplete Ni
2+
-chroma-
tography column (1 ml, Cat. no. 06781543001; Sigma-Aldrich), washed
in 20 CV buffer (50 mM Tris, pH: 9.0, 200 mM NaCl). Bound protein was
eluted with 250 mM imidazole. Following this, SEC was carried out on
a Superdex 75 16/60 HR column (GE Healthcare) equilibrated in 10
mM Tris, pH: 7.5, 200 mM NaCl.
Spy-2
Lysed cell pellets were homogenized and centrifuged at 20,000gfor 30
min. The filtered supernatant was loaded onto a Ni
2+
-chromatography
column(5ml;QIAGEN)andwashedin20CVbuffer(50mMTris,pH:8.5,
200 mM NaCl, 20 mM imidazole). Bound protein was eluted (in 50 mM
Tris, pH: 8.5, 200 mM NaCl, 250 mM imidazole), concentrated, and
subjected to SEC (Superdex 75 16/60 HR column; GE Healthcare).
Crystallisation, X-ray data collection, and structure
determination
Purified SRCR1 and SRCR8 were concentrated to 20 mg/ml. SRCR1 was
mixedwithanequalvolumeofmotherliquorcontaining0.2MMgCl
2
hexahydrate, 10% (wt/vol) PEG8000, 0.1 M Tris, pH: 7.0, and crystallised
in 400 nl drops by the vapor diffusion method at 21°C. SRCR8 was
mixed with an equal volume of mother liquor containing 0.1 M LiSO
4
,
20% (wt/vol) PEG6000, 0.01 M Hepes, pH: 6.5, and crystallised in 800 nl
drops. For SRCR8 + cation crystals were grown in 0.2 M MgCl
2
hexa-
hydrate, 20% (vol/vol) isopropanol, 0.1 M Hepes, pH: 7.5, and crys-
tallised in 400 nl drops. The crystallisation buffer was supplemented
with 10 mM Mg
2+
and 10 mM Ca
2+
, as well as 10 mM maltose, D-ga-
lactose, D-saccharose, D-mannose, D-glucose, and sucrose octa-
sulphate (all Sigma-Aldrich), 24 h prior to freezing. All crystals were
cryoprotected in mother liquor supplemented with 30% glycerol and
flashfrozeninliquidN
2
. Data were collected at a temperature of 80 K
Figure 5. SALSA scavenger receptor cysteine-rich (SRCR) cation-binding motif
reveals a conserved mechanism for broad-spectrum ligand interactions of
SRCR superfamily molecules.
Based on mutational studies and structural information across SRCR proteins, we
propose a generalised mechanism of ligand interaction mediated by the
cation-binding surface motif of the evolutionarily ancient SRCR fold (left side).
SALSA has been described to bind a broad range of ligands, incorporating into a
complex network of binding partners on the body surfaces and the colonizing
microbiota. The multiple SRCR domains of full-length SALSA bind repetitive
targets (both protein and carbohydrate structures) on the surface of microbes.
The secreted fluid-phase molecule may thus lead to microbial agglutination
and clearance. However, the repetitive form of binding sites will allow for
simultaneous binding to endogenous targets as well. This being, for example, 1)
binding of IgA, collectins, and complement components to induce a
cooperative antimicrobial effect; 2) binding of hydroxyapatite on the tooth
surface; and 3) ECM proteins and glycosaminoglycans, as well as mucus
components of the epithelial surface (such as heparin, galectin 3, and mucins)
(right side). The cation-binding motif described in SALSA is conserved in most
other SRCR proteins. For CD6, CD163, and MARCO, mutational studies support a
crucial role for this area in ligand interactions. CD163 binds the
haemoglobin–haptoglobin complex and microbial surfaces. CD6 binds
endogenous ligands but also engages in microbial binding. MARCO forms
multimers and binds microbial surface structures. Other SRCR proteins with
similar functions and conserved cation sites include SR-A1, Sp-α, SSc5D, and
M2bp. The functional role of the neurotrypsin SRCR domains is not known. The
remarkable repetitive formation of multiple SRCR domains in many SRCR
superfamily proteins, with several domains containing a binding site with a broad
specificity, would supposedly allow for interactions with multiple ligands
simultaneously. The SRCR fold thus appears to be an important functional
component of scavenging molecules engaging in complex network of
interactions. The multiple SRCR domains shown for SALSA, CD163, and
neurotrypsin are represented as copies of protein-specific SRCR domains with
known structure. Conserved cation-coordinating residues are highlighted in red.
SRCR8soak (light blue), MARCO (pdbid: 2oy3, sand), CD163 (pdbid: 5jfb, purple),
CD6 (pdbid: 5a2e, grey), M2bp (pdbid: 1by2, yellow), and murine neurotrypsin
(pdbid: 6h8m, teal).
SALSA domain structures Reichhardt et al. https://doi.org/10.26508/lsa.201900502 vol 3 | no 4 | e201900502 7of10
on beamlines I04, at a wavelength of 1.0718
˚
A (for SRCR1 and SRCR8)
and I03, at a wavelength of 0.9762
˚
A (for SRCR8cat) at the Diamond
Light Source, as specified in Table 1. The structure of SRCR8 was
solved by molecular replacement using MolRep within CCP4 (56) with
the structure of CD6 SRCR domain 3 (PDB ID 5a2e (27)). The structures
of SRCR1 and SRCR8 soaked in cations were solved by molecular
replacement using the structure of SRCR8. Refinement and re-
building were carried out in Phenix and Coot (57,58). Assignment of
metal ions was carried out by first refining the structure without
anything in the metal binding sites, followed by addition of com-
binations of probable ligands, and re-refinement in phenix.refine
using restraints generated by phenix.ready_set for each combina-
tion. The structures were characterised by the statistics shown in
Table 1 with no Ramachandran outliers. Protein structure figures
were prepared using Pymol version 2.0 (Schr¨
odinger, LLC).
Hydroxyapatite binding assay
150 μl hydroxyapatite nanoparticle suspension (Cat. no. 702153;
Sigma-Aldrich) was washed into buffer (10 mM Hepes, pH: 7.5, 150 mM
NaCl, 1 mM Ca
2+
). Beads were incubated in 80 μl SRCR8, SRCR8 D34A,
or SRCR8 D35A(all at 0.5 mg/ml inthe same buffer) with shaking for 1
h at RT. Beads were spun and washed 6× in 1 ml buffer. Bound protein
was eluted in 100 μl 0.5 M EDTA and visualized by SDS–PAGE (4–20%;
Bio-Rad) and Coomassie staining (Instant Blue, Expedeon).
Heparin binding assay
SRCR8, SRCR8 D34A, or SRCR8 D35A in 10 mM Hepes, pH: 7.5, 10 mM
NaCl, 1 mM Ca
2+
were loaded onto a HiTrap Heparin HP column (1 ml;
GE Healthcare), equilibrated in the same buffer. Bound protein was
then eluted with 10 mM Hepes, pH: 7.5, 10 mM NaCl, 20 mM EDTA.
Spy-2 binding assay
On a MaxiSorp plate (Nunc), 100 μl purified Spy-2 was coated O/N at
4°C in a concentration ranging from 0.032 to 3.2 μM in coating buffer
(100 mM NaHCO
3
buffer, pH: 9.5). The plate was blocked in 1% gelatine
in PBS, and SRCR8, SRCR8 D34A, and SRCR8 D35A were added (all at 7.1
μM in 10 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM Ca
2+
, 0.05% Tween20).
Boundproteinwasdetectedwithmonoclonalanti-SALSAantibody
diluted 1:10,000 (1G4; Novus Biologicals) and HRP-conjugated rabbit
anti-mouse antibody 1:10,000 (W4028; Promega). The plate was de-
velopedwith2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(Sigma-Aldrich) and analysed by spectrophotometry at 405 nm. To test
calcium-specific dependency of the interaction, the WT assay above
was repeated in a buffer containing 10 mM Hepes, pH 7.5, 150 mM NaCl,
1mMMg
2+
,1mMEGTA,and0.05%Tween20.
Data availability
Structure factors and coordinates from this publication have been
deposited to the PDB database https://www.wwpdb.org and assigned
the identifiers: SRCR1 pdbid: 6sa4, SRCR8 pdbid: 6sa5,SRCR8soakwith
three cations pdbid: 6san.
Supplementary Information
Supplementary Information is available at https://doi.org/10.26508/lsa.
201900502.
Acknowledgements
We acknowledge Diamond Light Source and the staff of beamlines I03 and I04
for access under proposal MX18069. The Central Oxford Structural Molecular
and Imaging Centre is supported by the Wellcome Trust (201536). MP Reich-
hardt was financially supported by grants from the Wihuri Foundation and the
Finnish Cultural Foundation. Staff andexperimental costs in SM Lealaboratory
were supported by a Wellcome Investigator Award (100298) and an Medical
Research Council (UK) programme grant (M011984). V Loimaranta was sup-
ported by the Turku University Foundation.
Author Contributions
MP Reichhardt: conceptualization, data curation, formal analysis,
funding acquisition, investigation, and writing—original draft, review,
and editing.
V Loimaranta: resources.
SM Lea: conceptualization, data curation, formal analysis, funding ac-
quisition, validation, investigation, methodology, project administration,
and writing—review and editing.
S Johnson: conceptualization, data curation, formal analysis, super-
vision, validation, investigation, visualization, methodology, project
administration, and writing—original draft, review, and editing.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
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