1
Unique DC-SIGN clus e ing ac i i y o a small glycomime ic: a lesson
o ligand design
Ie a Su ke iciu e
1,2,3
, Michel Thépau
1,2,3
, Sa a Sa in
4
, Angela Be zi
5
, John McGeagh
6
, Se gei
G udinin
7,8
, Jö g Weise
6
, Aline Le Roy
1,2,3
, Jose J. Reina
9
, Ja ie Rojo
9
, Ma io Cle ici
10
, Anna
Be na di
4
, Ch is ine Ebel
1,2,3
, F anck Fieschi
1,2,3*
1
Uni . G enoble Alpes, Ins i u de Biologie S uc u ale (IBS), G enoble, F-38027, F ance;
2
CNRS, IBS
G enoble, F-38000, F ance;
3
CEA, DSV-IBS, G enoble, F-38000, F ance;
4
Uni e si a’ di Milano,
Dipa imen o di Chimica, ia Golgi 19, 20133 Milano - I aly;
5
Depa men o Biomedical and Clinical
Sciences, Uni e si y o Milan, 20157 Milan, I aly;
6
An e io Consul &Resea ch GmbH, Augus aanlage 23, D-
68165 Mannheim-Ge many;
7
INRIA G enoble, Sain Ismie Cedex F-38334, F ance;
8
CNRS Labo a oi e
Jean Kun zmann, 38041 G enoble, F ance;
9
Glycosys ems Labo a o y, Ins i u o de In es igaciones
Químicas (IIQ), CSIC – Uni e sidad de Se illa, A . Amé ico Vespucio 49, Se ille 41092 Spain;
10
Depa men
o Physiopa ology and T ansplan a ion, Uni e si y o Milan, Milan, and Don C. Gnocchi Founda ion ONLUS,
IRCCS, 20148 Milan, I aly.
*
Co esponding au ho . E-mail: [email p o ec ed]; Tel: +33-(0)-4-57 42 85 23.
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Abs ac
DC-SIGN is a dend i ic cell-speci ic C- ype lec in ecep o ha ecognizes highly glycosyla ed ligands
exp essed on he su ace o a ious pa hogens. This ecep o plays an impo an ole in he ea ly s ages
o many i al in ec ions, including HIV, which makes i an in e es ing he apeu ic a ge . Glycomime ic
compounds a e good d ug candida es o DC-SIGN inhibi ion due o hei high solubili y, esis ance o
glycosidases and non- oxici y. We s udied he s uc u al p ope ies o he in e ac ion o he e ame ic
DC-SIGN ex acellula domain (ECD), wi h wo glycomime ic an agonis s, a pseudo-mannobioside (1)
and a linea pseudo-manno ioside (2). Though he inhibi o y po ency o 2, as measu ed by SPR
compe i ion expe imen s, was one o de o magni ude highe han ha o 1, c ys al s uc u es o he
complexes wi hin DC-SIGN ca bohyd a e ecogni ion domain showed he same binding mode o bo h
compounds. Mo eo e , when coupled o mul i alen sca olds, he inhibi o y po encies o hese
compounds became uni o m. Combining iso he mal i a ion mic ocalo ime y, analy ical
ul acen i uga ion and dynamic ligh sca e ing echniques o s udy DC-SIGN ECD in e ac ion wi h
hese glycomime ics, e ealed ha 2 is able, wi hou any mul i alen p esen a ion, o clus e DC-SIGN
e ame s leading o an a i icially o e es ima ed inhibi o y po ency. The use o mul i alen sca olds
p esen ing 1 o 2 in HIV ans-in ec ion inhibi ion assay con i ms he loss o po ency o 2 upon
coupling and he equal e icacy o chemically simple compound 1. This s udy documen s a unique
case whe e, among wo ac i e compounds chemically de i ed, he compound wi h he lowe appa en
ac i i y is he op imal lead o u he d ug de elopmen .
Keywo ds: C-Type Lec in, HIV, glycomime ics, clus e ing, analy ical ul acen i uga ion, iso he mal
mic ocalo ime y
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In oduc ion
Imma u e dend i ic cells (DCs) a e p o essional an igen p esen ing cells o he inna e immuni y.
They ou inely su ey pe iphe al issues o in ading pa hogens, and p esen he an igens o he T cells
hus boos ing pa hogen-speci ic adap i e immune esponses(1). Pa e n ecogni ion ecep o s (PRRs) o
DCs a e ins umen al in cap u ing pa hogens h ough he ecogni ion o pa hogen associa ed molecula
pa e ns (PAMPs). DCs exp ess a wide ange o PRRs including oll-like ecep o s (TLRs) and C- ype
lec in ecep o s (CLRs), enabling DCs o ecognize he majo i y o in ading pa hogens(2, 3). One o
hese PRRs is DC-SIGN (Dend i ic Cell-Speci ic ICAM-3 G abbing Non-in eg in), a CLR abundan ly
exp essed on imma u e DCs ha has been ex ensi ely s udied because o i s in iguing oles in
immuni y(4, 5). DC-SIGN was ecognized as a ecep o hijacked by a ious dange ous pa hogens,
including i uses like HIV and Ebola, bac e ia, ungi and pa asi es, o e ade o modula e hos immune
esponses enhancing hei in ec i i y(6, 7). These indings highligh ed DC-SIGN as an in e es ing
he apeu ic a ge and inspi ed many esea ch g oups o a emp he design o an agonis s o in ec ion
p e en ion.
DC-SIGN is a ype II memb ane p o ein wi h a cy osolic domain ollowed by a ansmemb ane
egion and an ex acellula domain (ECD). The la e is o ganized in o an elonga ed neck egion and a
globula C- e minal ca bohyd a e ecogni ion domain (CRD), which binds ucose and mannose-
con aining oligosaccha ides in a calcium-dependen manne (8, 9). The neck d i es lec in’s
oligome iza ion in o e ame s and se es as a s alk aising CRDs well abo e he cell memb ane
(320 Å) and p esen ing hem in a e a alen manne wi h ~40 Å dis ances be ween icinal binding si es
(10). These e ame s a e u he clus e ed o mic odomains a he cell memb ane(11, 12).
Al hough monosaccha ide a ini y o DC-SIGN is e y low, mul ime ic DC-SIGN o ganiza ion
oge he wi h clus e ed p esen a ion o pa hogen glycans leads o he a ini y enhancemen h ough
a idi y e ec s. Hence, he s a egies o de elop good DC-SIGN inhibi o s no only in ol e
op imiza ion o he mono alen ligands, bu also include ex ensi e sea ch o he p ope sca olds o
mul ime ic ligand p esen a ion, hus ensu ing e icien compe i ion wi h highly mul i alen PAMPs
( e iewed in
(13, 14)).
Ou g oups ocus on he de elopmen o ucose- and mannose-based glycomime ic inhibi o s o
DC-SIGN. We ecen ly published se ies o LewisX isaccha ide mimics, which had sligh ly be e
a ini y as compa ed o na u al LewisX and an imp o ed selec i i y o DC-SIGN e sus lange in(15), a
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C- ype lec in implica ed in he p o ec ion om HIV in ec ion(16). We ha e also de eloped wo
mannose- ype glycomime ics, co esponding o a e minal b anch o high-mannose glycan, which a e
he a ge ligands o DC-SIGN on he gp120 HIV en elope p o ein. Thus, we p oduced a Manα1-
2Man mimic pseudomannobioside (1) and a Manα1-2Manα1-6Man mimic pseudomanno ioside (2)
and e alua ed hem as DC-SIGN inhibi o s. The ini ial su ace plasmon esonance (SPR) compe i ion
assay indica ed ha 2 had an o de o magni ude lowe IC
50
han 1(17). Hence, his compound was
selec ed o gene a e a e a alen dend on 3.2, which in u n was es ed o he abili y o inhibi HIV
ans-in ec ion o T-lymphocy es media ed by B-THP-1/DC-SIGN cells(17) as well as HIV
ansmission inhibi ion in ce ical issue explan s(18). Indeed, he dend on 3.2 displayed a e y
p omising an i-HIV ac i i y in hese assays and was demons a ed o be non- oxic.
Addi ionally, he hi d gene a ion (G3) Bol o n ype dend ime s 4 bea ing an a e age o 30-32
copies o 1 (compound 4.1) o 2 (compound 4.2) we e buil and e alua ed in SPR compe i ion
expe imen s and DC-SIGN-media ed Ebola in ec ion assays(19). Bo h dend ime s we e highly ac i e
in inhibi ing Ebola in ec ion, bu su p isingly, no signi ican di e ence was ound in hei po encies
(IC
50
~20 nM o bo h compounds). The SPR compe i ion expe imen s ga e a simila ou come (Figu e
1): while he ac i i ies o he mono alen compounds ha e almos one o de o magni ude di e ence in
a o o 2, nea ly he same a ini ies we e obse ed o e a alen (3.1 and 3.2) and mul i alen (4.1
and 4.2) o ms o 1 and 2. Fu he mo e, he mono alen and e a alen o ms o 2 (2 and 3.2) had
basically he same ac i i ies.
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Figu e 1. The compa ison o 1 and 2 inhibi o y e iciencies a mono alen and mul i alen p esen a ions. (A) The s uc u es
o ligands 1-4; he ed ames highligh he po ions o he molecules conjuga ed o he sca olds; he s uc u e o G3
Bol o n ype dend ime 4, ep esen ed he e as a g ey sphe e, is shown in SI. (B) The IC
50
o he co esponding compounds
om SPR compe i ion assay, adap ed om e e ence (19).
Recen ly, we ha e s uc u ally cha ac e ized he binding o 1 o DC-SIGN CRD(20). X- ay
c ys allog aphy o co-c ys allized DC-SIGN CRD/1 complex e ealed an unexpec ed binding mode o
he molecule, which was con i med by solu ion s udies using ans e NOE ( -NOESY) and sa u a ion
ans e di e ence (STD) NMR expe imen s. Al hough 1 and i s na u al coun e pa Manα1-2Man
ha e simila a ini ies o DC-SIGN as obse ed in SPR compe i ion es (IC
50
alues 1.0 mM and
0.9 mM, espec i ely), ou esul s indica ed ha 1, con a y o Manα1-2Man, has only a single binding
mode wi hin DC-SIGN CRD, a p e equisi e o a good lead compound o u he chemical
modi ica ions o imp o e a ini y and selec i i y. We also ound ha 1 has an enhanced selec i i y
owa ds DC-SIGN compa ed o lange in. This is an impo an ea u e o he de elopmen o
mic obicides ha shouldn’ in e e e wi h he p o ec i e unc ion o lange in, especially due o i s
exp ession on Lange hans cells, which cons i u e he e y i s ba ie o in ading HIV i ions in
geni al and ec al mucosa(16).
He ein we p esen ou e o s o un a el he unde lying easons o he abo e-desc ibed
su p ising beha io o he compound 2. Th ough se ies o biophysical s udies we ound ha his small
glycomime ic, o ou su p ise, ac s as a bi alen molecule, and his peculia p ope y led o an a i icial
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o e es ima ion o i s po ency in compe i ion assays whe e soluble DC-SIGN e ame s a e used. This
wo k emphasizes he impo ance o ho ough and c i ical s uc u al in es iga ion o leads om p ima y
sc eening campaigns and highligh s speci ic design challenges o he disco e y o po en an agonis s
o lec ins’ ac i i y.
Resul s and discussion
The s udy desc ibed in his pape was mo i a ed by he need o unde s and why he ela i ely
high e iciency o 2 is los when he compound is e he ed on mul i alen sca olds, an essen ial
in o ma ion in o de o shape ou ligand design p og am. We ha e co-c ys allized he la e
glycomime ic wi h DC-SIGN CRD and sol ed he X- ay s uc u e. Fu he mo e, we in es iga ed he
he modynamic and hyd odynamic p ope ies o DC-SIGN ECD in e ac ion wi h 1 and 2 by iso he mal
i a ion mic ocalo ime y (ITC) and analy ical ul acen i uga ion (AUC). Ou esul s indica ed ha
mono alen 2 in ac unc ions as a bi alen molecule capable o b idging DC-SIGN e ame s, a e y
peculia p ope y o such a a he small molecule wi h ela i ely no so high a ini y. Such peculia
p ope y o 2 esul ed in an a i icial o e es ima ion o i s po ency in compe i ion assays whe e soluble
DC-SIGN e ame s a e used.
The X- ay s uc u e o DC-SIGN CRD/2 complex and i s compa ison wi h DC-SIGN CRD/1
In o de o compa e he binding modes o 1 and 2 wi hin DC-SIGN and hus o shed ligh on he
unde lying easons o he binding a ini y o mono- and poly alen e sions o 2, we co-c ys allized 2
wi h monome ic DC-SIGN CRD and sol ed he X- ay c ys al s uc u e.
Simila ly o he ecen ly published s uc u e o DC-SIGN/1, he c ys als o 2 in complex wi h
DC-SIGN CRD also had a single copy o CRD in an asymme ic uni wi h P4
3
2
1
2 space g oup. The
s uc u e was sol ed by molecula eplacemen , a 1.35 Å esolu ion (see Table S1 in SI).
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Figu e 2. The binding mode o pseudo-manno ioside 2 wi hin DC-SIGN CRD. (A) and (B) Two o ien a ions o he bound
ligand supe imposed wi h he Fo-Fc elec on densi y map (cyan 3σ
con ou ); he p o ein ca bon backbone is ep esen ed in
oli e ca oon wi h amino acids con ibu ing o binding highligh ed by s ick ep esen a ion; 2 is shown in yellow s icks.
V351 exhibi s wo al e na i e con o ma ions wi h 50% occupancy in he c ys al s uc u e, bo h o hem a e ep esen ed in
(B). (C) pseudo-manno ioside in e ac ion o DC-SIGN. Hyd ogen bonds a e shown as dashed pu ple lines, Ca
2+
coo dina ion bonds a e dashed black lines, and key an de Waals in e ac ion a e indica ed by dashed blue lines. (D) The
supe imposi ion o 1 (pink s icks) and 2 (yellow s icks) c ys al s uc u es bound o DC-SIGN CRD (pale cyan su ace
ep esen a ion). Oxygen and ni ogen a oms a e in ed and blue, Ca
2+
ion is a g een sphe e.
When he s uc u e was sol ed using he model wi hou suga , an elec on densi y was obse ed
on Ca
2+
ion in he canonical ca bohyd a e binding si e, indica ing he p esence o pseudomanno ioside
bound o DC-SIGN h ough a con en ional Ca
2+
-coo dina ion by he 3-OH and 4-OH g oups o he
non- educing end mannose moie y (Figu e 2A and 2B). I appea ed ha he binding mode o 2 wi hin
DC-SIGN CRD (Figu e 2A) was he same as ha o 1, wi h exac ly he same o ien a ion o he
common po ion o he molecule (Figu e 2C and 2D). To ou su p ise, no addi ional con ac s (o he
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han hose desc ibed p e iously o 1,pdb 2x 5) wi h he p o ein we e obse ed. While he non-
educing mannose moie y made coo dina ion bonds wi h Ca
2+
ion wi h i s 3-OH and 4-OH g oups and
C6 me hylene o he cyclohexane ing e ained he an de Waals con ac wi h Val351 side chain o he
p o ein, he educing-end mannose was o ien ed away om he p o ein and appa en ly didn’ make any
con ac s. Mo eo e , he elec on densi ies o his second mannose uni as well as o i s e hylazide linke
we e a he poo ly de ined, sugges ing a highe lexibili y o hese pa s (Figu e 2A). Thus he e ealed
s uc u e o 2 wi hin DC-SIGN didn’ p o ide any clue abou he eason why i could be a be e ligand
han 1 o DC-SIGN ha ing appa en ly he same binding mode and con ac s wi h he p o ein. This
in iguing inding p omp ed us o in es iga e he in e ac ion u he on.
The modynamic cha ac e iza ion o DC-SIGN in e ac ion wi h 1 and 2
To e alua e whe he posi i e en opy con ibu ions we e esponsible o he highe a ini y o
mono alen 2 ela i e o 1, we analyzed he in e ac ion o bo h glycomime ics wi h e ame ic DC-
SIGN by ITC. A he i s se , he same expe imen al condi ions wi h iden ical concen a ions o
in e ac ion componen s we e used. The pseudosaccha ides (12.7 mM) we e i a ed in o lec in solu ion
(71 µM wi h espec o binding si es).
Figu e 3. ITC esul s o 1 and 2 i a ions o DC-SIGN ECD. (A) and (B) show i a ions o 1 and 2, espec i ely, a
12.7 mM o DC-SIGN ECD (71 µM). (C) 2 (1.18 mM) i a ion o 214 µM DC-SIGN ECD. Uppe panels show he i a ion
he mog ams and lowe panels he da a in eg a ion wi h i ed cu es (1:1 binding model).
The ITC da a (Figu e 3A and 3B) indica ed he low a ini y o he ligands as he i a ion cu es
didn’ adop he ull sigmoidal shape. Fi ing one binding si e model o he da a wi h an assumed
s oichiome y alue n ixed o 1 yielded he K
D
o 990.10±19.7 µM and 75.76±7.29 µM o 1 and 2,
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espec i ely, bu he low a ini y p e en ed eliable in e ac ion en halpies and en opies o be ob ained
and compa ed(21). Ne e heless, an in iguing ou come could be obse ed: he i s injec ions o 2 o
DC-SIGN solu ion esul ed in wice mo e hea eleased using he same concen a ions o bo h ligands
and he ecep o . This sugges ed a highe a ini y o 2 han 1, which could no be explained by he X-
ay da a since 2 didn’ seem o make any o he addi ional con ac s compa ed o 1.
Because 2 had appa en ly highe a ini y o DC-SIGN, we epea ed he i a ion wi h di e en
concen a ions (1.18 mM o 2 i a ed o 214 µM o DC-SIGN ECD) in o de o pe o m i in a mo e
ele an concen a ion ange (Figu e 3C). Fi ing he same model his ime ga e K
D
o 5.26±0.29 µM,
∆H=-15.80±0.15 kcal mol
-1
, T∆S=-8.57 kcal mol
-1
, and ∆G o -7.20 kcal mol
-1
. Thus a di e ence o 2
o de s o magni ude be ween he K
D
alues o 1 and 2 was de e mined (990 µM and 5 µM,
espec i ely), which is much highe han wha could be ini ially expec ed om he SPR da a and
highligh s e en a s onge di e ence in he ac i i ies o he wo molecules.
Fu he mo e, i ing o he da a in Figu e 3C yielded n=0.5 s oichiome y. Because addi ional
DC-SIGN ECD pu i ica ion s ep has been applied o his expe imen (SI Figu e S2), he con ibu ion
o non-ac i e binding si es o n<1 alue was excluded. In e ac ion wi h n=0.5 in his case means ha
wo DC-SIGN CRDs sha e one molecule 2, o in o he wo ds, wo DC-SIGN e ame s bind ou
copies o 2, on a e age. Since 2 is a ela i ely small molecule, i is impossible o i o each wo
di e en CRDs wi hin he same e ame ( he app oxima e dis ance be ween icinal binding si es
wi hin he e ame is 40 Å(10)). Hence, his s oichiome y sugges s ha he same molecule o 2
b idges wo DC-SIGN e ame s by simul aneously binding o one CRD in each, and would explain
why unde he same expe imen al condi ions i a ing DC-SIGN wi h 2 eleased ma kedly mo e hea
han i a ing wi h 1.
Analy ical Ul acen i uga ion analysis o DC-SIGN/1 and DC-SIGN/2 complexes
To check he hypo hesis ha 2 can b idge DC-SIGN e ame s, sedimen a ion eloci y (SV)
expe imen s we e pe o med on he samples e ie ed om he ITC measu emen s, whe e he ligands
we e in 26- old excess ela i e o he lec in binding si es. Figu e 4 compa es he sedimen a ion p o iles
egis e ed a 42000 pm a he same ime in e als (1 h) o each o he h ee samples (pu e p o ein,
Figu e 4A; DC-SIGN+1, Figu e 4B; DC-SIGN+2, Figu e 4C). Clea ly, DC-SIGN alone o incuba ed
wi h 1 sedimen s simila ly, while DC-SIGN incuba ed wi h 2 mo es as e , sugges ing ha he
associa ion o DC-SIGN e ame s is induced by 2 bu no by 1.
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biological ele ance(28, 29), he case p esen ed he e is unique, conside ing he small size o he
molecule and he low a ini y o single mannose esidues o DC-SIGN. P obably, he absence o s e ic
hind ance be ween bo h DC-SIGN ECDs in he complex, as sugges ed by he modeling, allows such
b idging o occu . In biological sys ems he mos common si ua ion o mul i alen suga /p o ein
in e ac ions is he o ma ion o ca bohyd a e-lec in la ices(30) and lec in b idging p ope y o
glycomime ic molecules has been p e iously enginee ed in ela i ely la ge mul ime ic
p esen a ions(31-34). Howe e , o he bes o ou knowledge, b idging e ec s by an oligosaccha ide as
small as a (pseudo) ime ha e ne e been obse ed wi h one excep ion in he pa icula case o
c ys alliza ion condi ions(33).
Un o una ely, his p ope y o 2 depends on a p ecise o ma o he in e ac ion assay and
equi es bo h ligand and p o ein o be simul aneously in solu ion. This doesn’ co espond o he
biologically ele an si ua ion whe e he ecep o is ancho ed o cell memb ane and he ligand in
solu ion. Addi ionally, he b idging abili y o 2 appa en ly is los , e en in solu ion assays, when i is
e he ed o a mul i alen sca old. Thus his ligand canno ully p o i o a idi y e ec s gene a ed by
mul i alen p esen a ions. In ou de elopmen o DC-SIGN inhibi o s, whe e we aim o design
mul i alen pla o ms bea ing mul iple copies o mode a ely ac i e glycomime ic leads, he b idging
abili y o 2 was he unde lying eason o i s o e es ima ion in compe i ion assays. Once his ligand is
e he ed o mul i alen sca olds, he second mannose moie y ac s basically as an ex ended linke
p o iding no ad an ages. Wi hou a ho ough s uc u al cha ac e iza ion o he binding mode o 1 and 2
o DC-SIGN ( e e ence (20) and his wo k, espec i ely), compound 2 would ha e been chosen o
u he chemical op imiza ion on he sole basis o IC
50
esul s. This op ion would ha e been ex emely
cos ly, on a syn he ic poin o iew, and p obably ine ec i e a he end. F om he ou come o hese
in e ac ion analyses, i is clea ha op imiza ion o he mono alen ligand, he e o e, should ocus on
he s uc u e o he pseudo-disaccha ide 1, which is syn he ically much simple han 2. Ou ecen
imp o emen s in his di ec ion ha e been ecen ly epo ed(35). Besides p o iding a s uc u al a ionale
o obse a ions made du ing ou DC-SIGN an agonis s esea ch p og am, hese esul s deli e a
gene al lesson o he design o lec in an agonis s.
Me hods
Compound syn hesis.
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Syn heses o 1(36), 2, 3.2(17) 3.1, 4.1 and 4.2(19) we e desc ibed p e iously.
DC-SIGN p oduc ion and pu i ica ion
DC-SIGN CRD-S epTagII (S-CRD) and ECD cons uc s we e p oduced as desc ibed p e iously(10,
20).
DC-SIGN/2 co-c ys alliza ion, da a collec ion and s uc u e solu ion
C ys alliza ion was pe o med a 20°C in EasyX al pla es (Qiagen) by hanging-d op apo -di usion
me hod. The d ops we e p epa ed combining 1 µL o a pu i ied DC-SIGN S-CRD (4.4 mg mL
-1
in
25 mM T is-HCl pH 8, 150 mM NaCl, 4 mM CaCl
2
, bu e -A) and 2 (300 mM) mix u e (9:1, / ) wi h
1 µL o ese oi solu ion and equilib a ed agains 1 mL o ese oi solu ion. The bes c ys als we e
ob ained when ese oi solu ion was 35% PEG3350, 100 mM cacodyla e pH 6.5, and 200 mM NaCl.
The X- ay di ac ion da a collec ion, s uc u e solu ion and e inemen a e desc ibed in Supplemen a y
In o ma ion. C ys al s uc u e o DC-SIGN CRD/2 has been deposi ed in P o ein Da a Bank unde
PDB code 2x 6.
Iso he mal i a ion mic ocalo ime y
ITC expe imen s we e pe o med a 25°C using Mic ocal/VP-ITC mic ocalo ime e (Mic ocal,
No hamp on, MA) wi h 1.4 mL cell olume. . The pseudosaccha ides and DC-SIGN ECD we e
p epa ed in bu e -A. Pseudosaccha ides we e s epwise injec ed (10 µL aliquo s) o DC-SIGN solu ion
using 5 min in e als be ween injec ions. In he i s se o expe imen s 71 µM (in e ms o binding
si es) lec in and 12.7 mM pseudosaccha ide concen a ions we e used; in he second se 1.18 mM 2 was
i a ed o 214 µM DC-SIGN ECD. The blank i a ions (compounds o bu e ) we e done o
sub ac ion o dilu ion hea om he in eg a ed da a. A one-si e binding model was i o he da a
(Mic ocal O igin-7) yielding associa ion cons an s (K
A
) and binding en halpies (∆H). The ee ene gy
changes (∆G) and en opy (∆S) we e calcula ed using equa ion:
∆G = ∆H − T∆S = −RT ln K
A
whe e T is he absolu e empe a u e, and R=8.314 J mol
−1
K
−1
.
S a ic and Dynamic Ligh Sca e ing
The SLS and DLS we e pe o med using DynaP o/Nanos a ins umen (Wya Technology Co p.,
San a Ba ba a, USA) equipped wi h 658 nm lase a 90° sca e ing angle. T iplica e measu emen s o
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10 sca e ing eadings pe sample in a qua z cu e e we e eco ded a 25°C. The samples, p epa ed in
bu e -A and cen i uged p io o analysis, con ained DC-SIGN ECD alone (102 µM wi h espec o
binding si es) o wi h 2 a di e en 2/DC-SIGN mola a ios (0, 0.11, 0.25, 0.5, 1, 5, 11 and 27). The
da a we e analyzed wi h Dynamics-7.1 so wa e (Wya Technology Co p.).
Analy ical Ul acen i uga ion
SV expe imen s we e pe o med using Beckman XL-I analy ical ul acen i uge wi h AN-50 TI o o
(Beckman ins umen s), a 20°C and 42 000 pm, using 55 µl samples, loaded in o he wo-channel
0.15 cm pa h-leng h cen e pieces wi h sapphi e windows (Nanoly ics). The abso bance a 280 nm was
moni o ed e e y 5 o 11 min o he i s and second se expe imen s, espec i ely, wi h a 30 µm adial
s ep size.
The samples we e p epa ed in bu e -A. The i s se o expe imen s was done wi h samples e ie ed
om i s se ITC measu emen s, hus con ained DC-SIGN ECD alone a 62 µM, o wi h 1.63 mM 1 o
2. The iden ical eshly p epa ed samples we e used o he con ol, gi ing iden ical esul s.
Fo he second se , he samples con ained 102 µM DC-SIGN ECD alone o wi h 2 added a di e en
2/DC-SIGN mola a ios (0, 0.25, 0.5, 1, 5, 11 and 27). The MW and pa ial speci ic olume ( ) o
DC-SIGN e ame s we e es ima ed om amino acid composi ion using SEDNTERP so wa e and
esul ed 154827 Da and 0.733 cm
3
g
-1
, espec i ely. The - alues o 1 and 2 we e conside ed equal o
- alue o a hexose (0.607 cm
3
g
-1
). SV p o iles we e analyzed using he size dis ibu ion analysis(39)
embedded in he SEDFIT so wa e (a ailable a h p://www.analy icalul acen i uga ion.com), and o
each analysis by global modeling aking ypically 20 egula ly spaced expe imen al p o iles ob ained
o e 4 h. The c(s) analysis was pe o med conside ing 200 pa icles, and i ing he ic ional a io, /
0
.
Fo he egula iza ion, a 0.68 con idence le el was used. All s- alues we e co ec ed o sol en densi y
and iscosi y, and hus a e gi en as s
20w
- alues.
DC-SIGN/2 b idging complex modeling.
To gene a e a e ahed al DC-SIGN assembly, we used a no el symme y docking algo i hm(25). We
assembled a e ahed al oc ame possessing D4 symme y s a ing om a monome o he epo ed
c ys al s uc u e (PDB 2x 6) wi h a single mannose in he Ca
2+
binding-si e using he exhaus i e sea ch
in six deg ees o eedom. Then, we selec ed an oc ame ic solu ion ha ul illed geome ic cons ain s
a he coiled–coil s a and i ed he pseudo isaccha ide 2 in o i s binding pocke using Au odock/Vina
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so wa e(40). Solu ions, whe e ECD o ganiza ion was compa ible wi h he p e iously desc ibed DC-
SIGN ECD SAXS en elope, ha e been conse ed(10). To se -up he ligand docking simula ions, we
used Au odock/Vina plugin o PyMOL(41).
Calcula ion o heo e ical hyd odynamic adius om he model.
HYDROPRO p og am(26) was used o ob ain he heo e ical sedimen a ion coe icien alue o he
model o b idged DC-SIGN e ame s. The expe imen al alues o sol en iscosi y, densi y and
empe a u e we e η=0.01024 poise, ρ=1.006 g cm
-3
, and he empe a u e was 293 K. The esul ing
sedimen a ion coe icien s- alue o 6.848 S was used o calcula e heo e ical R
H
using S edbe g’s
equa ion:
,
whe e MW he molecula mass o he dime o DC-SIGN e ame s (309.654 kDa), and is he pa ial
speci ic olume o he dime o e ame s (0.733 cm
3
g
-1
).
In ec ion s udies.
The s udies o HIV ans-in ec ion inhibi ion by 3.1, 3.2, 4.1 and 4.2 wi hou emo al o compounds
p io o i us exposu e we e pe o med as desc ibed p e iously(17).
Acknowledgmen s:
This wo k was suppo ed by he Ma ie Cu ie ITN FP7 p ojec CARMUSYS (PITN-GA-2008-213592)
and he Agence Na ionale de la Reche che (ANR-11-MONU-006-01). A. Be zi was suppo ed by a
ellowship o UNIMI and Regione Lomba dia (P og amma “Do e Rice ca- Ra o za e il capi ale
umano”, POR-Ob.2 Asse IV-FSE 2007-2013). We a e g a e ul o P . Anne Imbe y and D . Ayme ic
Aud ay o an access o ITC ins umen and o hei kind suppo and ad ice. FF hanks he Ins i u
Uni e si ai e de F ance o inancial suppo . This wo k has been conduc ed hanks o he access o he
MP3, AUC and PAOL pla o ms o he Pa ne ship o S uc u al Biology and he Ins i u de Biologie
S uc u ale in G enoble (PSB/IBS).
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