Structural basis for the broad specificity to host-cell
ligands by the pathogenic fungus Candida albicans
Paula S. Salgadoa,1, Robert Yana,1,2, Jonathan D. Taylora, Lynn Burchella,3, Rhian Jonesa, Lois L. Hoyerb,
Steve J. Matthewsa, Peter J. Simpsona, and Ernesto Cotaa,4
aDivision of Molecular Biosciences, Imperial College London, Exhibition Road, South Kensington SW7 2AZ, United Kingdom; and
Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802
Edited by Ralph R. Isberg, HHMI/Tufts University School of Medicine, Boston, MA, and approved August 2, 2011 (received for review March 3, 2011)
Candida albicans is the most prevalent fungal pathogen in humans
and a major source of life-threatening nosocomial infections. The
Als (agglutinin-like sequence) glycoproteins are an important viru-
lence factor for this fungus and have been associated with binding
of host-cell surface proteins and small peptides of random se-
quence, the formation of biofilms and amyloid fibers. High-resolu-
tion structures of N-terminal Als adhesins (NT-Als; up to 314 amino
acids) show that ligand recognition relies on a motif capable of
binding flexible C termini of peptides in extended conformation.
Central to this mechanism is an invariant lysine that recognizes
the C-terminal carboxylate of ligands at the end of a deep-binding
cavity. In addition to several protein–peptide interactions, a net-
work of water molecules runs parallel to one side of the ligand
and contributes to the recognition of diverse peptide sequences.
These data establish NT-Als adhesins as a separate family of pep-
tide-binding proteins and an unexpected adhesion system for
primary, widespread protein–protein interactions at the Candida/
NMR ∣ X-ray crystallography ∣ microbial adhesion ∣ tissue tropism
Candida species, with Candida albicans the most frequent causa-
tive agent. Adhesion of the fungus to host surfaces correlates
positively with the pathogenic potential of different Candida spe-
cies (1). These adhesive properties manifest in different forms,
such as attachment to different cell types, binding to specific pro-
teins on the host cell surface and in the ECM, autoaggregation,
and the development of biofilms. Although many Candida cell-
wall proteins have been identified, molecular mechanisms of
interaction with the host remain poorly understood. Among the
most-studied C. albicans proteins implicated in this property are
the Als (agglutinin-like sequence) cell-surface glycoproteins (2).
Eight genetic loci encode Als proteins that are designated Als1 to
Als7 and Als9; ALS9 alleles encode proteins with distinct N-term-
inal domains (Als9-1 and Als9-2) (3). Als proteins are composed
of a signal peptide, an N-terminal region with 41–84% identities
among sequences in C. albicans, a nonrepeat Thr-rich (TR) re-
gion of 103aa., a central region with a variable number of 36aa.
repeats, and a Ser/Thr/Asn-rich C-terminal domain of variable
length that attaches the protein to the cell wall via the remnant
of a GPI anchor (3). Extensive N- and O-glycosylation are ob-
served in the central and C-terminal domains (4). Unlike the
highly glycosylated nature of the central and C-terminal domains
of Als proteins, the N-terminal region is predicted to form an
Ig-like structure and has been directly implicated in the binding
of cell surfaces (5).
Heterologous expression of individual ALS genes is sufficient
to confer upon Saccharomyces cerevisiae the adhesive/aggregative
properties observed in C. albicans (6), and cell-based experiments
suggest that Als proteins bind a range of host-cell and ECM
proteins including fibronectin, laminin, casein, BSA, type IV col-
lagen, cadherins, and ferritin (7–10). Furthermore, C. albicans or
otherwise nonadherent S. cerevisiae expressing Als1 and Als5
reakdown in host immunity or alterations in the normal
microbiota are common precursors to disease caused by
can efficiently bind 0.1–10% of beads loaded with a library of
107random heptapeptides (11).
Previous studies raise two important questions: How can Als
proteins recognize such a structurally diverse set of ligands, and
how is this activity related to the broad tissue tropism displayed by
C. albicans? For a detailed description of the binding mechanism
of these proteins, we solved the crystal structures of the 33-kDa
N-terminal (NT) region from Als9-2 in the presence and absence
of ligands. A preliminary solution structure of NT-Als1 and bind-
ing analyses of these adhesins were also obtained by NMR.
NT-Als Adhesins Adopt an Ig-Like MSCRAMM (Microbial Surface Com-
ponents Recognizing Adhesive Matrix Molecules) Architecture. We
have determined the structure of NT-Als9-2 in complex with a
peptide from the C-terminal end of human fibrinogen gamma,
Fg-γ (NH3-GEGQQHHLGGAKQAGDV-CO2). The structure
shows that NT-Als adhesins comprise two tandem DEv-IgG type
immunoglobulin domains (12), N1 and N2 (Fig. 1), arranged in a
fold reminiscent of MSCRAMM domains, as those in bacterial
adhesins ClfA from Staphylococcus aureus (13) and SdrG from
Staphylococcus epidermidis (14) (Fig. S1). Notable structural
differences include the presence of four conserved intradomain
disulfide bonds, an α-helix in the interdomain region (α1), and a
small β-strand essential for ligand binding (C1?) (Fig. S2), but the
relative domain–domain orientation is retained, as judged by the
rmsd of ca. 5 Å between NT-Als9-2 and these MSCRAMMs
(Fig. S1), plus residual dipolar coupling and small-angle X-ray
scattering (SAXS) data (Fig. S3).
NT-Als Adhesins Have a Cavity to Bind Peptides with Broad Specificity.
Details of the interactions of NT-Als9-2 with the Fg-γ peptide are
described in Fig. 1 (an electron density map is shown in Fig. S4).
In this complex, the ligand binds in a cavity formed by βG2b
and βA2 and is covered by the long A1-B1 loop from domain N1.
Author contributions: P.S.S., R.Y., J.D.T., S.J.M., P.J.S., and E.C. designed research; P.S.S., R.Y.,
J.D.T., L.B., R.J., and P.J.S. performed research; L.L.H. contributed new reagents/analytic
tools; P.S.S., R.Y., J.D.T., L.B., R.J., S.J.M., P.J.S., and E.C. analyzed data; and P.S.S., R.Y.,
L.L.H., S.J.M., P.J.S., and E.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 2Y7L, 2Y7N, and 2Y7O).
1P.S.S. and R.Y. contributed equally to this work.
2Present address: MRC National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, United Kingdom.
3Present address: Cancer Research UK, Lincoln’s Inn Fields Laboratories, London WC2A 3LY,
4To whom correspondence may be addressed at: Room 502, Biochemistry Building,
Imperial College London, South Kensington SW7 2AZ, United Kingdom. E-mail: e.cota@
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1103496108PNAS ∣ September 20, 2011 ∣ vol. 108 ∣ no. 38 ∣ 15775–15779
A cluster of conserved, bulky side chains defines the end of this
binding cleft and prevents the peptide from crossing the inter-
domain region and reaching N1 (Fig. 1B). The bound peptide
also makes extensive interactions with conserved hydrophobic
side chains (Y21, L295, W297, and Y301). Notably, the side chain
amine of an invariant lysine (K59) at the end of the cavity estab-
lishes a salt bridge with the C-terminal carboxylic acid of the
incoming peptide (Fig. 1B). This interaction explains the prefer-
ence for binding free peptide C termini and sets theinitial register
for the parallel hydrogen bonding pattern between the ligand
and βG2b (Fig. 2). To our knowledge, the specific recognition
of the C-terminal carboxylate is an exclusive property of NT-Als
adhesins relative to other families of peptide-binding proteins
(PBPs) (15, 16).
Apart from these structural features, NT-Als adhesins depend
on additional interactions to bind peptides with broad specificity.
Analysis of sequence conservation shows that surface variability is
mainly localized in the vicinity of the ligand, i.e., at positions that
induce changes in the shape and chemical properties of the bind-
ing cavity (β-strands A2, B2, F2a/b, G2a/b, and the A1-B1 loop)
(Fig. 3). In common with other PBPs, contacts with the ligand are
also aided by water-mediated hydrogen bonds. A network of
water molecules allows a malleable interaction between βA2 and
the side chains of different peptides, whereas their backbones
adopt a β-strand parallel to βG2b (Fig. 2 and Table S1). Small
rearrangements in the flexible A1-B1 loop also contribute to
the observed broad peptide-binding specificity.
Ligand Binding Studies of NT-Als Adhesins. NT-Als1 and NT-Als9-2
are monomeric in solution, as judged by size exclusion chroma-
tography, SAXS, and NMR relaxation experiments (Figs. S3
and S5). Surprisingly, however, in the absence of peptides we
observe that these adhesins recognize their own C termini as
ligands, as illustrated in the crystal structure of NT-Als9-2, in
which the C-terminal end is found in the binding cavity of a neigh-
boring adhesin (Fig. 4A and Fig. S6A). Similarly, in an attempt to
reduce the flexibility of this C terminus, we tested if introduction
of a bulky side chain in a cavity at the interdomain region could
induce the conformation of βG2′ observed in the NT-Als9-2/Fg-γ
complex. Unexpectedly, the crystal structure of the G299W mu-
tant shows that the C-terminal end folds back to self-complement
its own binding site (Fig. 4B and Fig. S6B). Using NMR, a com-
NT-Als9-2 in complex with Fg-γ peptide (NH3-GEGQQHHLGGAKQAGDV-
CO2); underlined residues correspond to modeled peptide (red), as identified
in the electron density (Fig. S4A). Structural elements involved in ligand bind-
ing are highlighted: β-strands A2 and G2b (yellow), G2′ (purple), loop A1-B1
(cyan), and a small β-strand containing K59 (dark blue). (B) Representation of
key residues involved in ligand binding (F293, L295, W297, yellow spheres)
and hydrophobic residues at the end of the binding cavity (V19, P29, I61,
F58 and Y301, purple spheres). A dashed line shows the interactions between
K59 and the C-terminal end of the peptide. The aminoacid numbering across
the text is based on the sequence alignment of C. albicans NT-Als adhesins
shown in Fig. S2. The C-terminal valine of human fibrinogen-γ (V437) is also
Structure of NT-Als adhesins. (A) Cartoon representation of
number of residues observed in the binding pocket of NT-Als adhesins is in agreement with the minimal peptide size recognized by Als proteins (11). A network
of water molecules (spheres, cyan) mediates interactions between peptide and protein. The A1-B1 loop (cyan) creates a flexible cap to the binding cavity.
(B) Same as A, rotated approximately 90° on the x axis. (C) Fg-γ peptide specific interactions with NT-Als9-2 are represented as dark red dashed lines, whereas
interactions conserved in all complexes described in this paper are indicated by gray dashed lines. Conserved K59 is colored in dark blue. In these complexes,
hydrogen bond interactions are between protein and ligand atoms, including those mediated by bound water molecules. C was produced using
Detailed view of peptide interactions with NT-Als9-2. (A) View of the peptide-binding cavity of NT-Als9-2 with bound Fg-γ peptide (sticks, red). The
www.pnas.org/cgi/doi/10.1073/pnas.1103496108Salgado et al.
parable effect is observed in NT-Als1: We have identified NOEs
(nuclear Overhauser effects) between residues in the binding
cavity and the C-terminal residues V314 and A315, indicative that
the binding pocket of this adhesin is also complemented in
solution by its own C terminus via intra- or interdomain interac-
tions (Fig. 4C and Fig. S7). This is confirmed by NMR dynamics
measurements (T1, T2relaxation and heteronuclear NOE) for
backbone amides of NT-Als1, which show that C-terminal resi-
dues I311–A315 have relaxation rates comparable to the folded
part of the protein, whereas connecting residues D305–G310 are
largely unstructured, i.e., exhibiting faster internal motion on the
nanosecond–picosecond time scale (Fig. S5A).
It is important to emphasize that the C-terminal end of NT-Als
is not available for these interactions in vivo because the NT
domain is only one portion of the much larger Als molecule.
Similarly, the very C-terminal end of the full-length Als protein
is not available for these interactions in vivo because it is attached
by a GPI anchor remnant to the Candida cell wall. As a result, the
self-complemented structures do not reflect the true conforma-
tion of the C-terminal residues of NT-Als adhesins in the apo
form. However, they suggest that this region is flexible in the ab-
sence of ligands. Self-complementation by endogenous NT-Als
C-terminal sequences (which were selected arbitrarily for produc-
tion of folded, soluble NT-Als fragments) supports the conclusion
that Als adhesins have a broad specificity for peptide binding.
Upon binding of Fg-γ peptide, the C-terminal end of NT-
Als9-2 extends away from N2 to form the new strand (βG2′) over
N1, resembling the ligand bound conformation of bacterial
MSCRAMMs (Fig. 1A). Also notable is the change in orientation
of conserved Y301: Solvent-exposed in the self-complemented
forms, it flips 180° to form a hydrogen bond with the buried K59.
The presence of the peptide also induces alterations in the con-
formation of the A1-B1 loop, now in contact with the ligand and
flanked by the newly formed C-terminal strand, rendering it less
flexible, as indicated by better defined electron density and lower
B factor values (Fig. S5B).
To test whether these interactions can be displaced by free
peptides in solution, we recorded two-dimensional NMR spectra
of15N labeled NT-Als1 and 9-2 titrated with a threonine hepta-
mer (NH2-TTTTTTT-CO2) and the Fg-γ peptide, respectively.
In both cases, a large number of chemical shift changes occur
upon titration, which indicates that binding induces a structural
transition between free and bound forms (Fig. 5A and Fig. S8A).
In NT-Als1, the largest shift changes map to residues in the
binding pocket (A19 and K59), the C terminus (I308 and A311),
and others that interact with βG2′ (e.g., D72 and K106) (Fig. 5B),
in a pattern consistent with the conformation of the ligand bound
crystal form of NT-Als9-2. Simultaneous disappearance and
appearance of peaks from free and bound conformations, respec-
tively, is indicative of binding in the “slow exchange regime” on
the NMR time scale, as shown for the backbone amide of A315
from NT-Als1 (Fig. S8B). Assuming a diffusion-controlled on
rate (kon), this result suggests dissociation constants KDsmaller
than approximately 10 μM (17). In these conditions, no chemical
shift changes were observed on titration of NT-Als1 with the Fg-γ
peptide or NT-Als9-2 with the threonine heptamer.
An Invariant Lysine (K59) and a Free C-Terminal Carboxylate Are Essen-
tial for Ligand Binding. In agreement with the observation of a salt
bridge between K59 and the ligand C-terminal carboxylate in dif-
ferent crystallographic complexes, C-terminal amination of pep-
tides is sufficient to abolish binding, as indicated by an absence of
protein shift changes after addition of up to 100 equivalents of
ligand. Similarly, cocrystallization of NT-Als9-2 with C-terminally
aminated Fg-γ peptide results in crystals where only the apo form
is identifiable. Interestingly, proton NMR spectra demonstrate
that the K59M and K59V mutants in NT-Als9-2 are sufficient
to induce folding defects in these adhesins, suggesting that inter-
actions with this residue might also be involved in protein stability
(Fig. S8C). A similar analysis was not possible for NT-Als9-2
K59R, K59E, and K59A mutants, as they induce reduced protein
yields in our Escherichia coli expression system and aggregation
after protein purification.
The conformation of NT-Als9-2 in complex with the Fg-γ peptide
is reminiscent of the proposed “dock, lock, and latch” mechanism
described for the binding of SdrG and ClfA to their specific
ligands (18). However, the mode of ligand recognition of Als
proteins is distinct. In bacterial MSCRAMMs, the peptide-bind-
ing site is distributed on both Ig-like domains, leaving the N- and
C termini of the ligand exposed to the solvent. In contrast, the
peptide-binding mechanism for Als proteins is inconsistent with
the recognition of unstructured interdomain/loop regions or the
showing the values representing surface conservation of C. albicans NT-Als
adhesins, in a gradient where the most conserved residues are shown in
white and the least conserved in dark blue. (B) Surface representation of
A. (C) Surface representation with a 180° rotation relative to B. The ligand
is shown as a red wire, for reference. Residue conservation values were
obtained using ConSurf (40).
Sequence conservation in NT-Als adhesins. (A) Cartoon of NT-Als9-2
G299W mutant (B), the solution structure of NT-Als1 (C) and the crystal structure of NT-Als9-2 in complex with Fg-γ (D) are shown. The color coding is as in Fig. 1.
Details of the preliminary structure determination of NT-Als1 by NMR are presented in Fig. S7.
Changes in the conformation of C-terminal G2′ β-strand in purified NT-Als adhesins. Cartoons of the crystal structures of wild-type NT-Als9-2 (A) and
Salgado et al.PNAS
September 20, 2011
surface of folded domains: The binding cavity in the N2 domain
is limited to contain up to six residues of the ligand, whereas the
requirement to neutralize the buried charge of K59 provides
specificity for the C termini of incoming unstructured peptides.
Importantly, this mechanism is supported by published observa-
tions that Als proteins are able to bind a large range of structu-
rally unrelated proteins and peptides from randomly generated
Despite the ability to recognize a range of unrelated substrates,
there are documented differences in ligand specificity between
Als proteins (10, 11), potentially due to variations in C-terminal
peptide sequences that can be accommodated in the binding
clefts of their respective N-terminal adhesins. Our structural data
suggest that in vivo, these adhesins identify overlapping subsets
of C-terminal sequences on the surface of host cells and the ECM
(Fig. 6 A and B). Association to different ligands is likely en-
hanced by avidity effects from multiple interactions in the micro-
bial host–cell interface and increased expression of Als proteins,
as in the case of Als1 and Als3 (19).
Moreover, genes from a family of 10 secreted aspartyl protei-
nases (Sap1-10) are differentially expressed by C. albicans during
infection (20). Genomic transcriptional profiling shows a compar-
able up-regulation pattern for Als1, Als3, and Sap5 (with maxi-
mal expression levels at 3 h) in an intraperitoneal mouse model
(21). Intriguingly, Saps have been directly implicated in Candida
adherence (22), but the molecular mechanism underlying their
involvement in these events has not been established. In this
scenario, it is conceivable that proteolysis could increase Als-
mediated adhesion by creating additional C termini as potential
new ligands (Fig. 6 C–E); alternatively, proteolytic peptides re-
leased by the action of Saps might inhibit Als-mediated adhesion.
In addition to the peptide-binding mechanism described,
C. albicans adhesion has been associated to the aggregative prop-
erties of regions beyond the NT-Als domains, the amyloidogenic
and glycosylated sequences involved in the formation of biofilms
(23, 24). In this regard, it has been shown that the TR region
adjacent to the NT-Als domains contains a conserved amyloido-
genic motif (25), important for hydrophobic clustering and the
formation of “nanoadhesomes” (26). The NT-Als9-2/Fg-γ peptide
complex shows that this region (-GIVIV- in Als1 and 5) is part of
the C-terminal βG2′ that docks over the surface of N1, i.e., pre-
ceding the TR region. Interestingly, substitution of V312 to N
(GIVIV) in Als5 decreases formation of amyloid fibrils in vitro,
as a purified protein or expressed on the yeast cell surface (27).
The structural transitions of this region on the surface of C. albi-
cans and their functional implications remain to be determined.
More recently it has been shown that a fragment encompassing
residues 18–432 of Als1 recognizes different fucose-containing
sugars from a glycan array (28). The authors also demonstrate that
BSA has a dissociation constant in the submillimolar range
(KD¼ 210 μM), which opens the possibility that Als proteins can
specifically recognize fucose-related glycans or glycoproteins in
the peptide-binding cavity or an alternative binding site.
Crucially, the peptide-binding properties of NT-Als adhesins
provide a promiscuous mode of molecular recognition, as they
overcome the strict “domain-to-domain” surface complementar-
ity observed in most interactions between microbial adhesins
and host-cell receptors. Our structural data pave the way to study
the intriguing idea that changes in Candida physiology can be pro-
moted by the interplay of Als and other fungal cell-wall proteins
of Als-based anti-Candida vaccines (30). Conserved features in
the deep-binding cavity of these adhesins suggest a template for
the design of drugs that block key interactions between Candida
and host cells.
relaxation-optimized spectroscopy–heteronuclear sequential quantum corre-
lation (TROSY-HSQC) NMR spectra of NT-Als1. Peaks represent backbone
and side-chain amides of the protein in the absence (black) and presence
of 50 molar equivalents of NH2-TTTTTTT-CO2peptide (red). (B)15N amide
chemical shifts observed in the surface of NT-Als1 upon ligand binding.
Changes in δ1H and δ15N from the spectra above were normalized as de-
scribed elsewhere (41). Ten different shades of white to red correlate with
increasing chemical shift changes.
Peptide binding in NT-Als1 observed by NMR. (A)1H15N transverse
domains of Als (ellipsoids) protrude from the Candida cell wall (A) and bind
proteins in the surface of host cells and the extracellular matrix (B). (C) So-
luble Saps (in green) and membrane bound Saps (in orange) partially digest
proteins in the host cell surface (D) and provide additional free C termini,
enhancing the Als-mediated adhesion process (E).
Model for Als-mediated interactions in vivo. The apical N-terminal
www.pnas.org/cgi/doi/10.1073/pnas.1103496108 Salgado et al.
Materials and Methods Download full-text
We expressed and purified the 33-kDa N-terminal adhesins of Als1 and
Als9-2 in E. coli as described (31, 32). The Quikchange mutagenesis kit
(Agilent Technologies) was used to generate NT-Als9-2 mutants G299W,
K59 to M, V, R, A, and E.
Crystallographic Data Collection, Processing and Structure Refinement. For
NT-Als9-2 crystals in the peptide free form, a dataset to 2.0-Å resolution
was collected in beamline BM14, European Synchrotron Research Facility, at
100 K, wavelength 0.9785 Å, using MARmosaic 225 detector with 1º oscillation
per image and processed using HKL2000 (33) (see Table S3 for details). Diffrac-
tion data for NT-Als9-2 crystals with bound peptide to 1.5 Å were collected at
100 K in beamline I03, Diamond Light Source, using an ADSC (Area Detector
Systems Corporation) 315r detector with 1º oscillation per image and pro-
cessed using HKL2000, as detailed in Table S3. For NT-Als9-2 G299W, data were
collected at PROXIMA1 beamline, Synchrotron SOLEIL, with a wavelength of
0.9919 Å, and data were processed using XDS (34). For all crystal forms, initial
phases were calculated with Phaser (35) using a refined Pt-derived NT-Als9-2
model (31), within the CCP4 program suite (36). Iterative cycles of model build-
ing and refinement were carried out using COOT (37) and REFMAC5 (38),
respectively, to determine the final models.
Binding and Relaxation Analyses of NT-Als1 by NMR. For observation of
chemical shift changes upon ligand binding,15N NT-Als1 (concentrated to
200 μM in 50 mM sodium phosphate pH 6.0, 50 mM NaCl, 10% D2O) was
titrated with 0.75, 2, 7.5, 20 and 50 M equivalents of a threonine heptamer
equivalents of Fg-γ peptide (Fig. S8). Changes were monitored by1H15N
TROSY-HSQC NMR spectra recorded in a 600-MHz magnet at 308 and 303 K
for NT-Als1 and NT-Als9-2, respectively.
15N NT-Als9-2 (150 μM) was titrated with 100 M
ACKNOWLEDGMENTS. We thank beamline staff for technical assistance and
support, particularly K. McAuley at Diamond Light Source, V. Olieric at the
Swiss Light Source, A. Thompson and P. Legrand at Synchrotron SOLEIL,
H. Belrhali at BM14 beamline, European Synchrotron Radiation Facility,
and D. Svergun and M. Roessle at X33-DESY beamline, as well as mem-
bers of the E.C. and S.J.M. labs at Imperial College London for generous
support. The authors also acknowledge J. Moore and the Centre of Struc-
tural Biology at Imperial College London. This work was funded by Grant
R01 DE14158 from the National Institute of Dental and Craniofacial Re-
search, National Institutes of Health (to L.L.H.) and by the Biotechnology
and Biological Sciences Research Council, Project Grant BB/F007566/1
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