Structure of the Complex between a Heparan Sulfate Octasaccharide and Mycobacterial Heparin-Binding Hemagglutinin

Article abstract of DOI:10.1002/anie.201612518

Heparin-binding hemagglutinin (HBHA) is a 199 amino acid virulence factor at the envelope of Mycobacterium tuberculosis that contributes to latent tuberculosis. The binding of HBHA to respiratory epithelial cells, which leads to extrapulmonary dissemination of the pathogen, is mediated by cell-surface heparan sulfate (HS). We report the structural characterization of the HBHA/HS complex by NMR spectroscopy. To develop a model for the molecular recognition, the first chemically synthesized uniformly ¹³C- and ¹⁵N-labeled HS octasaccharide and a uniformly ¹³C- and ¹⁵N-labeled form of HBHA were prepared. Residues 180–195 at the C-terminal region of HBHA show large chemical shift perturbation upon association with the octasaccharide. Molecular dynamics simulations conforming to the multidimensional NMR data revealed key electrostatic and even hydrophobic interactions between the binding partners that may aid in the development of agents targeting the binding event.

Full text of DOI:10.1002/anie.201612518

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International Edition: DOI: 10.1002/anie.201612518
German Edition: DOI: 10.1002/ange.201612518
Protein NMR
Structure of the Complex between a Heparan Sulfate Octasaccharide
and Mycobacterial Heparin-Binding Hemagglutinin
Teng-Yi Huang+, Deli Irene+, Medel Manuel L. Zulueta, Tzu-Jui Tai, Shih-Han Lain, Cheng-
Po Cheng, Ping-Xi Tsai, Shu-Yi Lin, Zhi-Geng Chen, Chiao-Chu Ku, Chwan-Deng Hsiao, Chia-
Lin Chyan,* and Shang-Cheng Hung*
Dedicated to Professor Chun-Chen Liao on the occasion of his 75th birthday
Abstract: Heparin-binding hemagglutinin (HBHA) is a 199
amino acid virulence factor at the envelope of Mycobacterium
tuberculosis that contributes to latent tuberculosis. The binding
of HBHA to respiratory epithelial cells, which leads to
extrapulmonary dissemination of the pathogen, is mediated
by cell-surface heparan sulfate (HS). We report the structural
characterization of the HBHA/HS complex by NMR spec-
troscopy. To develop a model for the molecular recognition, the
first chemically synthesized uniformly 13C- and 15N-labeled HS
octasaccharide and a uniformly 13C- and 15N-labeled form of
HBHA were prepared. Residues 180–195 at the C-terminal
region of HBHA show large chemical shift perturbation upon
association with the octasaccharide. Molecular dynamics
simulations conforming to the multidimensional NMR data
revealed key electrostatic and even hydrophobic interactions
between the binding partners that may aid in the development
of agents targeting the binding event.
domain (residues 12–29), a coiled-coil domain (residues 29–
109), and a C-terminal region (residues 160–199) rich in
lysine, proline, and alanine residues.[4] The coiled-coil domain
acts as a trigger sequence for protein dimerization and
promotes bacterial agglutination.[5]
HS is a polysaccharide component of proteoglycans found
on cell surfaces and in the extracellular matrix.[6] It holds
alternating (1!4)-linked a-d-glucosamine (GlcN) and either
b-d-glucuronic acid (GlcA) or a-l-iduronic acid (IdoA)
residues. O- and N-sulfonations occur to variable extents,
which, together with the carboxylate group, provide a poly-
anionic chain and a complex functional-group pattern.[7]
These properties facilitate association with many circulating
proteins, including growth factors, chemokines, and microbial
proteins such as HBHA.[8] Heparinase treatment[9] and anti-
HBHA monoclonal antibodies[10] disrupt the attachment of
HBHA to epithelial cells. A deep understanding of this
recognition process could thus inspire new therapeutic
strategies against tuberculosis.
Electrostatic forces likely feature prominently in the
interactions between HS and HBHA, as observed with many
other HS-binding proteins.[6] To investigate the binding
process and avoid heterogeneous isolates from natural
sources, we previously synthesized HS oligosaccharides bear-
ing 2-N- and 6-O-sulfonated GlcN and 2-O-sulfonated
IdoA.[11] Such units are abundant in the sulfated regions of
HS and are also the major components of heparin, a related
sugar that is mainly localized within mastocytes. Using these
compounds, the hexasaccharide was identified as the mini-
mum length required for binding to HBHA. The octasac-
charide analogue provided a dissociation constant (KD) of
4.2 mm in an entropically driven 1:1 interaction with a trun-
cated form of HBHA (i.e., HBHA60-199), thus curiously
suggesting that hydrophobic interactions could be major
contributors to the binding. Mindful that the seemingly
unstructured nature of the C-terminal region[5a] may deter
X-ray characterization, we turned to NMR spectroscopy to
probe the complexation process at the molecular level. To
address the severe resonance overlaps that have historically
hindered NMR interpretations, the first chemical synthesis of
a uniformly 13C- and 15N-labeled HS octasaccharide (here-
after referred to as HS8) was carried out. Similar uniform
labeling was also implemented on a form of HBHA expressed
in Escherichia coli with 13C-labeled glucose and 15NH4Cl as
the sources of carbon and nitrogen atoms, respectively.
T
uberculosis is a major global threat with over a million
annual deaths and a high incidence of drug resistance.[1] The
causative agent, Mycobacterium tuberculosis, invades alveolar
macrophages and can survive and evade the host defenses. It
is a terrible pulmonary disease in active form, but most
infections lead to latent tuberculosis with no clinical expres-
sion.[2] Critical for enabling latency is heparin-binding hemag-
glutinin (HBHA). This mycobacterial envelope protein is
essential for binding to heparan sulfate (HS) at the surface of
respiratory epithelial cells, thereby promoting extrapulmo-
nary dissemination.[3] HBHA contains a transmembrane
[*] Dr. T.-Y. Huang,[+] Dr. M. M. L. Zulueta, Dr. C.-P. Cheng, Dr. P.-X. Tsai,
Dr. S.-Y. Lin, Dr. Z.-G. Chen, Prof. Dr. S.-C. Hung
Genomics Research Center, Academia Sinica
No. 128, Section 2, Academia Road, Taipei 115 (Taiwan)
E-mail: schung@gate.sinica.edu.tw
Dr. D. Irene,[+] T.-J. Tai, S.-H. Lain, Prof. Dr. C.-L. Chyan
Department of Chemistry, National Dong Hwa University
No. 1, Section 2, Da Hsueh Road, Shoufeng, Hualien 974 (Taiwan)
E-mail: chyan@gms.ndhu.edu.tw
C.-C. Ku, Prof. Dr. C.-D. Hsiao
Institute of Molecular Biology, Academia Sinica
No. 128, Section 2, Academia Road, Taipei 115 (Taiwan)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201612518.
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To prepare the desired labeled HS8, a uniformly 13C-
labeled d-glucose (1) was transformed into the crucial
anhydro-l-idose and GlcN building blocks 3 and 8, respec-
tively (Scheme 1). Both 3 and 8 were designed to offer
excellent stereoselectivity during glycosylations and permit
the final functionalizations.[11,12] The synthesis of 3 began with
Scheme 1. Preparation of the monosaccharide building blocks. Aster-
isks indicate 13C- or 15N-labeled atoms. Bn=benzyl, Bz=benzoyl,
NAP=2-naphthylmethyl, PBB=p-bromobenzyl, TBDPS=tert-butyldi-
phenylsilyl, Tf=triflyl, TMS=trimethylsilyl, Tol=p-tolyl.
Scheme 2. Preparation of the uniformly 13C- and 15N-labeled HS8.
Ac=acetyl, BAIB=bis(acetoxy)iodobenzene, DDQ=2,3-dichloro-5,6-
dicyano-1,4-benzoquinone, NIS=N-iodosuccinimide, TBAF=tetra-
butylammonium fluoride, TEMPO=2,2,6,6-tetramethyl-1-piperidinyl-
oxyl free radical.
the formation of the diacetonide 2. The transformation then
followed the typical method[13] of C5-epimerization and 1,6-
anhydroidose formation, thereby ultimately generating the
desired 4-alcohol 3. Introducing the 15N label is the major
concern for the GlcN synthesis. Inspired by an early report,[14]
we accessed this unit through epoxide 5. After considering
some 15N sources such as azide (undesirable 1,3-dipolar
property) or phthalimide (less than satisfactory yield), we
settled on 15N-labeled benzylamine for epoxide opening
under Sc(OTf)3 catalysis. Subsequently, the resulting 15N-
labeled amine 6 was converted into the azide 7. Simultaneous
thioglycosylation and anhydro-ring opening, followed by 6-O-
silylation led to the fully functionalized thioglycoside 8.
We then shifted to oligosaccharide assembly and func-
tional-group transformations (Scheme 2). Activation of thio-
glycoside 8 in the presence of 3 supplied the key disaccharide
9, which was transformed into the corresponding acceptor 10
and thioglycoside donor 11 for the next round of glycosyla-
tion. Convergent [2+2] and [4+4] glycosylations effectively
generated the fully protected octasaccharide 15. Partial
deblocking, selective oxidation, O-sulfonation, global depro-
tection, and final N-sulfonation delivered the target analogue
16 (labeled HS8).
increased by 6%, roughly equivalent to a 12-residue a-helical
structure (Table S1 in the Supporting Information). Con-
versely, no noteworthy changes occurred in the CD spectra
when HS8 was added to the truncated forms HBHA60-199
,
HBHA110-199, and HBHA1-160 (the subscripts are amino acid
positions). The thermodynamic parameters for the complex-
ation of HS8 with HBHA87-199, HBHA110-199, and HBHA160-199
were also acquired by ITC (Figure S2 and Table S2). As with
HBHA60-199,
[11] the interactions were endothermic and largely
stabilized by entropic factors, with comparable KD values in
the low micromolar range (3.18–7.46 mm). HS8 has a binding
stoichiometry of around 1:1 with both HBHA87-199 and
HBHA110-199, but a 1:2 sugar-to-protein binding ratio was
recorded with HBHA160-199. These data indicate that HS8
binds to the C-terminal region and induces a conformational
change in HBHA, increasing the helical content within
residues 1–60.
With adequate water solubility and reasonable affinity to
HS8, HBHA110-199 was chosen as a surrogate in our NMR
characterization. The 13C- and 15N-labeled HBHA110-199 ena-
bled resonance assignments of the free protein through
a series of multidimensional NMR experiments (see the
Supporting Information).[16] Aliphatic side-chain proton and
carbon chemical shifts were assigned by through-bond
correlations and then confirmed by nuclear Overhauser
Circular dichroism (CD) spectroscopy and isothermal
titration calorimetry (ITC) gave preliminary insight into the
complexation process. Addition of HS8 to full-length HBHA
changed the CD absorptivities at 208 and 222 nm (Figure S1
in the Supporting Information). Analysis[15] showed that, at
the expense of some b-sheet residues, the a-helical content of
full-length HBHA in the presence of equimolar HS8
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effects (NOEs) in 13C- and 15N-separated NOE spectroscopy
(NOESY)-heteronuclear single quantum correlation (HSQC)
spectra. A similar analysis for bound HBHA110-199 was
performed using a sample containing labeled HBHA110-199
and unlabeled HS8. The chemical shift index algorithm[17]
confirmed that free HBHA110-199 is mostly unstructured, and
no secondary structure was induced upon association with
HS8. The solution structures of free and bound HBHA110-199
were calculated by CYANA.[18] The majority of the NOE
cross-peaks were from intra-residue and sequential proton
pairs, and medium-range NOEs were found in three regions,
indicating short a-helical structures (within residues 116–120,
128–138, and 146–150).
Chemical-shift perturbation was used to map the inter-
action site and to calculate KD by regression analysis. The
NMR spectra of a series of complexes of labeled HBHA110-199
with unlabeled HS8 in different stoichiometric ratios were
acquired, taking note of the perturbed cross-peaks of
HBHA110-199 (Figure 1a and Figure S3). The weighted chem-
ical-shift perturbation for each residue (Ddresidue) was calcu-
lated (see the Supporting Information). The residues K180,
A181, A182, A183, K185, A186, K190, A191, A192, A193,
K194, and K195 were the most perturbed, with Ddresidue values
more than three times the average (0.019 ppm). Situated near
the end of the C-terminal region of HBHA, these residues are
likely in proximity to the interaction surface or undergo major
structural readjustment upon binding. The close match of the
perturbed amino acids with that of a previous NMR analysis
utilizing a 14-mer heparin sample and a chimeric protein[4b]
indicates their pivotal roles. The cross-peak movements with
increasing concentrations of HS8 suggest fast ligand exchange
(Figure 1b and Figure S4). Analysis of the tracked cross-
Figure 1. Perturbations in HBHA110-199 upon complexation with HS8.
A) The weighted chemical-shift perturbation (Ddresidue) of HBHA110-199
upon association with HS8 plotted against residue number. The yellow
line corresponds to the average Ddresidue (0.019 ppm); two- and
threefold averaged Ddresidue are indicated in magenta and cyan,
respectively. B) Overlaid 1H-15N HSQC spectra showing the movement
of the cross-peaks of key residues of labeled HBHA110-199 upon titration
with unlabeled HS8. The spectrum of free HBHA110-199 is shown in red,
with cyan for the complex of HS8 with HBHA110-199 in a stoichiometric
ratio of 0.5, orange for a ratio of 1, and in blue for a ratio of 2.
peaks shows that HS8 has 1:1 stoichiometry with HBHA110-199
,
with an average KD of 4.49 mm (Table S3), which is consistent
with ITC data.
Resonance assignments of free and bound HS8 were
obtained by correlating intra- and inter-residue through-bond
and through-space connectivities (Figure S5–S8). The NMR
spectra for bound HS8 were acquired from a sample contain-
ing labeled HS8 and unlabeled HBHA110-199. The chemical
shifts of residues A, F, and G are well-dispersed, whereas
residues B and C are degenerate to residues D and E,
respectively (Table S4, see compound 16 in Scheme 2 for
sugar residue notation). The majority of the HS8 cross-peaks
show only minor or no deviation upon association with
HBHA110-199. Coupling constants (Table S5) were used to
elucidate the ring conformations[19] and to derive the dihedral
angles according to the Karplus correlation.[20] While all of the
GlcN residues were in the 4C1 conformation, the IdoA
residues appeared as mixtures of 2SO and 1C4 in free
HS8.[19–21] Upon interaction with HBHA, a preference for
the 2SO conformation was observed.[22] This was confirmed by
the strong NOE intensities between H2 and H5 of the IdoA
residues in the bound form; the corresponding free-form
NOEs were much weaker (Figure S8). Consistent with
restricted motion, more NOEs were found for bound than
free HS8.
and dihedral angle restraints from the NMR data (Table S6–
S9). Focusing on the lysine e proton and the alanine b proton,
16 intermolecular NOE restraints were deduced from the 13C-
separated NOESY–HSQC spectra of the doubly labeled
HBHA110-119 and HS8 (Table S10). Twenty lowest-energy
structures from the simulations, which fulfil the binding
interface defined by the intermolecular NOEs and chemical-
shift deviation experiments, were selected (Figure 2a). The
best low-energy structure was chosen for further analysis. In
this model, the protein formed three prominent loops
spanning residues 181–185, 186–189, and 193–199 in such
a way as to produce a shallow binding ridge for HS8. As
expected, lysine residues, specifically, K179, K180, K184,
K185, K189, and K199, participated in ionic interactions with
the sulfonate and carboxylate groups found within residues
A–G of HS8 (Figure 2b). Notably, the terminal K199 residue
looped around and formed salt bridges with the 2-O-sulfonate
group of D (IdoA), the 6-O-sulfonate group of E (GlcN), and
the carboxylate group of F (IdoA) residues. Hydrogen
Structural modeling of the sugar/protein complex was
performed using restrained molecular dynamics with distance
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water molecules in the sugar to create the entropic effect that
drives the binding process. On the other hand, the protein
residues that showed highly perturbed chemical-shift values
but were not involved in interacting with the sugar (A183,
K190, K194, and K195) instead underwent structural rear-
rangement of the flexible random coil to permit the binding of
HS8 to HBHA.
In summary, we have examined the complexation of HS8
with HBHA using CD, ITC, and NMR spectroscopy. The
NMR analysis was enhanced by the use of 13C- and 15N-
labeled forms of both the sugar and the protein, thereby
providing key information that was beneficial in the molec-
ular dynamics simulation of the complex. We noted relevant
electrostatic interactions between the lysine side chains and
the sulfonate and carboxylate groups of the sugar. The
hydrophobic interactions that we suspected to be major factor
in the binding were also clarified as coming from certain
lysine aliphatic regions and alanine residues populating the C-
terminal region. Overall, these molecular details should offer
important insight into the binding process and present new
opportunities for drug design against latent tuberculosis.
Further examination of the binding process and its effect on
the conformation of HBHA is in progress.
Acknowledgements
This work was supported by the Ministry of Science and
Technology of Taiwan (MOST 104-2113-M-259-006, MOST
104-0210-01-09-02, MOST 104-2628-M-001-001, MOST 105-
0210-01-13-01, and MOST 105-2745-M-001-003-ASP),
National Dong Hwa University, and Academia Sinica.
Figure 2. Modeling of the HBHA/HS8 complex, focusing on the
interaction site. A) Superposition of the 20 lowest-energy conformers
of HBHA and HS8. B) Intermolecular ionic interactions (magenta
dashed line) and hydrogen bonding (orange dashed line) with HBHA
shown as a transparent surface. C) Hydrophobic interactions involving
K179, A181, A186, A191, and A193 with the relevant atoms shown as
transparent spheres. HS8 is shown with cyan carbon atom; HBHA
carbon atoms and backbone are shown in dark gray. The rest of the
model has the color scheme N blue, O red, S yellow, H white.
Conflict of interest
The authors declare no conflict of interest.
Keywords: carbohydrates · heparan sulfate ·
heparin-binding hemagglutinin · NMR spectroscopy ·
protein structures
bonding, mainly through the 6-sulfate group of the E residue
of HS8, also helped anchor the sugar to the protein. Judging
from the length and pattern of the lysine-rich C-terminal
region, a sugar length about twice that of HS8 would probably
be accommodated by HBHA. Moreover, the minimal
requirement of six sugar units (represented by residues C to
H) found by ITC[11] also made sense in this model.
Hydrophobic interactions were implied by the entropi-
cally driven binding of HS8 to HBHA. Our model shows that
four alanine residues participate in hydrophobic clusters with
the sugar rings (Figure 2c and Figure S9). A181 and A186
associate with the B-D rings of the sugar, whereas A191 and
A193 engage in close interaction with E-H rings. The key
presence of several alanines alongside multiple lysine residues
within the HS-binding C-terminal region of HBHA provides
an opportunity for this notable hydrophobic interaction to
occur.[8] In addition, the aliphatic side-chain component of
K179 shows hydrophobic interactions with the A-residue
(GlcN) atoms of HS8. These interfaces likely displace ordered
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Manuscript received: December 24, 2016
Revised: February 11, 2016
Final Article published: && &&, &&&&
[16] The complete 1H, 13C, and 15N chemical-shift assignments the
free and bound HBHA110-199 have been deposited in the
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