RESEARCH ARTICLE
S udy o he in e ac ions o bo ine se um
albumin wi h a molybdenum(II) ca bonyl
complex by spec oscopic and molecula
simula ion me hods
He
´lia F. Je emias
1☯
, Diana Lousa
1☯
, Axel Hollmann
2
, Ana C. Coelho
1
, Ca la S. Bal aza
1
,
João D. Seixas
3
, Ana R. Ma ques
3
, Nuno C. San os
2
, Ca los C. Romão
1,3
*, Cla
´udio
M. Soa esID
1
*
1ITQB NOVA, Ins i u o de Tecnologia Quı
´mica e Biolo
´gica An o
´nio Xa ie , Uni e sidade No a de Lisboa,
Oei as, Po ugal, 2Ins i u o de Medicina Molecula , Faculdade de Medicina, Uni e sidade de Lisboa, Lisbon,
Po ugal, 3Al ama Lda., Ins i u o de Biologia Expe imen al e Tecnolo
´gica, Oei as, Po ugal
☯These au ho s con ibu ed equally o his wo k.
*[email p o ec ed] (CMS); [email p o ec ed] (CCR)
Abs ac
The apy wi h inhaled ca bon monoxide (CO) is being es ed in human clinical ials, ye he
al e na i e use o p od ugs, CO-Releasing Molecules (CORMs), is concep ually ad an a-
geous. These molecules a e designed o elease ca bon monoxide in speci ic issues, in
esponse o some locally exp essed s imulus, whe e CO can igge a cy op o ec i e
esponse. The design o such p od ugs, mos ly me al ca bonyl complexes, mus conside
hei ADMET p o iles, including hei in e ac ion wi h anspo plasma p o eins. Howe e ,
he molecula de ails o his in e ac ion emain elusi e. To shed ligh in o his ma e , we
ocused on he CORM p o o ype [Mo(η
5
-Cp)(CH
2
COOH)(CO)
3
] (ALF414) and pe o med a
de ailed molecula cha ac e iza ion o i s in e ac ion wi h bo ine se um albumin (BSA),
using spec oscopic and compu a ional me hods. The expe imen al esul s show ha
ALF414 pa ially quenches he in insic luo escence o BSA wi hou changing i s seconda y
s uc u e. The in e ac ion be ween BSA and ALF414 ollows a dynamic quenching mecha-
nism, indica ing ha no s able complex is o med be ween he p o ein T p esidues and
ALF414. The molecula dynamics simula ions a e in good ag eemen wi h he expe imen al
esul s and con i m he dynamic and unspeci ic cha ac e o he in e ac ion be ween
ALF414 and BSA. The simula ions also p o ide impo an insigh s in o he na u e o he
in e ac ions o his CORM p o o ype wi h BSA, which a e domina ed by hyd ophobic con-
ac s, wi h a con ibu ion om hyd ogen bonding. This kind o in o ma ion is use ul o u u e
CORM design.
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 1 / 15
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Ci a ion: Je emias HF, Lousa D, Hollmann A,
Coelho AC, Bal aza CS, Seixas JD, e al. (2018)
S udy o he in e ac ions o bo ine se um albumin
wi h a molybdenum(II) ca bonyl complex by
spec oscopic and molecula simula ion me hods.
PLoS ONE 13(9): e0204624. h ps://doi.o g/
10.1371/jou nal.pone.0204624
Edi o : L. Michel Espinoza-Fonseca, Uni e si y o
Michigan, UNITED STATES
Recei ed: June 29, 2018
Accep ed: Sep embe 11, 2018
Published: Sep embe 27, 2018
Copy igh : ©2018 Je emias e al. This is an open
access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
a ailable om he Open Science F amewo k (os .
io/3ms47).
Funding: This wo k was inancially suppo ed by
Fundac¸ão pa a a Ciência e a Tecnologia, Po ugal,
h ough p ojec s PTDC/QUI-BIQ/117799/2010 and
Pes -OE/EQB/LA0004/2011. This wo k was also
inancially suppo ed by P ojec LISBOA-01-0145-
FEDER-007660 (Mic obiologia Molecula ,
Es u u al e Celula ) unded by FEDER unds
In oduc ion
The cy op o ec i e ac i i y o induced heme oxygenase (HO-1) is a well- ecognized p ope y
o his enzyme, which is induced o des oy oxic, ee heme, libe a ed om inju ed o dead
cells, he eby p oducing Fe
2+
ions, bili ubin and ee ca bon monoxide (CO)[1]. The la e is a
e y impo an signaling molecule (o en called gaso ansmi e ), akin o NO and H
2
S[2,3].
Impo an ly, applying CO om an ex e nal sou ce elici s essen ially he same physiological
cy op o ec i e esponses as HO-1, namely as an an i-in lamma o y, an i- h ombo ic and an i-
p oli e a i e agen .[4] This sugges s ha CO can be used as a he apeu ic, as indeed demon-
s a ed in animal models o ascula and in lamma o y diseases, ansplan a ion and a ange o
o he indica ions[5,6]. Howe e , he ansla ion o his po en ial in o eal, p ac ical CO-based
he apy aces many hu dles[7].
The na u al abili y o hemoglobin (Hb) o sca enge CO s ongly limi s he use o CO inha-
la ion as a use ul o m o ea men . Mo eo e , al hough used in animal models and human
clinical ials, such me hod lacks issue speci ici y and may equi e he use o high ( oxic) CO
concen a ions in sys emic ci cula ion o p oduce biological e ec s[8]. The e o e, a emp s o
ci cum en his di icul y ha e been es ed using p od ugs known as CORMs (CO-Releasing
Molecules). Mos CORMs a e ansi ion-me al ca bonyl complexes (MCCs), which deca bo-
nyla e in i o mo e easily han o he o ganic unc ions[9]. Ideally, CORMs mus su i e in ci -
cula ion and a ge he o gan in need, whe e hey elease CO in esponse o some locally
exp essed igge [10–12]. Once i en e s ci cula ion, he MCC in e ac s wi h plasma p o eins.
O hese, he mos abundan is se um albumin (SA) which unc ions as an impo an ans-
po e o bo h endogenous and exogenous molecules in ci cula ion,[13] hus de e mining se -
e al aspec s o he pha macokine ics o d ugs[14,15].
The in e ac ion o MCCs wi h SA may ha e h ee limi ing scena ios: 1) he in e ac ion
in ol es ex ensi e me al-ligand subs i u ion accompanied by libe a ion o CO; 2) me al-ligand
exchange is ex ensi e bu CO elease is limi ed o much slowe ; 3) he in e ac ion is non-co a-
len and CO is e ained wi hou bond b eaking o bond o ma ion. The i s case is exempli ied
by [Mo(his idina e)(CO)
3
]Na (ALF186), which apidly eleases CO o he blood s eam and
does no show Mo-HEWL (HEWL = Hen Egg Whi e Lysozyme) co alen in e ac ions in
model soaking expe imen s[16]. The second si ua ion is bes exempli ied by he eac ion o
[Ru(CO)
3
Cl(glycina e)] (CORM-3) wi h bo h HEWL o BSA. In bo h cases one equi alen o
CO is eleased as CO
2
, and adduc s o [Ru(CO)
2
]
2+
wi h he p o eins a e o med. The adduc
BSA-Ru(CO)
2
[17,18], was con i med o be esponsible o he slow deli e y o CO o a ge s in
i o[18]. O he examples a e gi en by he o ma ion o HEWL-M
I
(CO)
3
(M
I
= Mn
+
, Re
+
)
om p o ein and [L
3
M(CO)
3
]
+
complexes.[19,20] Such me al-ligand subs i u ion eac ions
a e simila o hose obse ed wi h an i-cance me al-based d ugs de oid o CO ligands.[21] In
he hi d si ua ion, he non-co alen in e ac ion o he MCC wi h he p o ein elies on in e -
molecula o ces, including, in some cases, hyd ogen bonds. In he absence o chemical eac-
ions, such MCCs esis decomposi ion in he blood and do no inc ease COHb.
Expe ience ells us ha M-CO bonds and o ganome allic ing ligands like π-cyclopen adie-
nyl (π-Cp) impa ma ked lipophilic cha ac e o he MCCs. The e o e, hey should in e ac
wi h hyd ophobic p o ein si es. Howe e , such in e ac ions a e s ill no e y well cha ac e -
ized. In ac , ine o ganome allic complexes a e no el ools in Chemical Biology, whe e hey
ha e been success ully used as enzyme inhibi o s and luminescen p obes[22]. Al hough hei
in e ac ions wi h speci ic enzyme binding si es ha e been s udied, we a e no awa e o any
s udy on he in e ac ions o o ganome allic ine CORMs wi h plasma anspo p o eins.
In o ma ion om such s udies is impo an o he design o ine CORM p od ugs in e ms
o he p edic ion o hei pha macokine ic and ADMET pa ame e s. Indeed, o be use ul and
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 2 / 15
h ough COMPETE2020 - P og ama Ope acional
Compe i i idade e In e nacionalizac¸ão (POCI) and
by na ional unds h ough FCT - Fundac¸ão pa a a
Ci00EAncia e a Tecnologia. DL was suppo ed by
FCT pos -doc ellowship SFRH/BPD/92537 The
unde Al ama Lda, owned by Al ama Inc. p o ided
suppo in he o m o sala ies o au ho s [CCR,
JDS, ARM] as well as cos s o he chemis y
[JDS] and biological esea ch [ARM] and ini ial
spec oscopic esea ch a IMM (NCS, AH]. JDS
and ARM le Al ama Lda in 2011 when i closed all
esea ch ac i i ies. CCR esumed his academic
posi ion a ITQB in July 2011 and emained as p o-
bono manage o Al ama Lda and Boa d membe
o Al ama Inc un il i was acqui ed by P o e is Inc
in 2017, becoming P o e is (Po ugal) Lda. This
esea ch was con inued a ITQB [CCR, ACC, HFJ
spec oscopy; CMS, DL, HFJ, CSB, compu a ional
s udies] and IMM [NCS, AH, spec oscopic
s udies] using exis ing ALF414 s ock samples and
exclusi ely inanced by Fundac¸ão pa a a Ciência e a
Tecnologia, Po ugal [p ojec PTDC/QUI-BIQ/
117799/2010 and Pes -OE/EQB/LA0004/2011] as
well as ITQB ins i u ional unds. Since 2011 nei he
Al ama Lda no P o e is Inc played any u he
inancial, scien i ic o comme cial ole in his
esea ch, which was ca ied ou a he academic
le el wi h speci ic. The unde s had no ole in s udy
design, da a collec ion and analysis, decision o
publish, o p epa a ion o he manusc ip .
Compe ing in e es s: CCR was co- ounde and
manage o Al ama Lda and Boa d membe o
Al ama Inc un il 2017. CCR is a s ockholde o
P o e is Inc. P o e is Inc does no ha e any
es ic ions on sha ing he p esen da a and
adhe es o he PLOS ONE policies on sha ing da a
and ma e ials.
o accomplish hei he apeu ic unc ion, he in e ac ions o he p od ug wi h SA mus be well
balanced. I hey a e oo s ong, he CORM may ail o exchange SA o he a ge disease issue
o may ail o eac wi h he chemical o enzyma ic igge s equi ed o CO elease [23,24]. I
hey a e oo weak, CORM’s hal -li e in ci cula ion may dec ease due o easy exc e ion o o
de oxi ica ion happening be o e i is able o deli e i s CO load.
In o de o ob ain undamen al in o ma ion on he na u e o such in e ac ions, we selec ed
[Mo(π-Cp)(CH
2
COOH)(CO)
3
] (ALF414; π-Cp = π-C
5
H
5
= cyclopen adienyl)(Fig 1) as a
model compound ea u ing a π-Cp ing, a subs i u ed me hyl ligand wi h a Mo-C σ-bond and
h ee CO ligands. [25]. Adequa ely o ou pu poses, i s globula 3D s uc u e p esen s wo
hyd ophobic egions, he a oma ic π-Cp ing and he CO ligands, as well as a me alloca -
boxylic ligand ha suppo s pola and H-bonding in e ac ions. ALF414 does no unde go
apid he mally induced subs i u ion eac ions, excep a high pH. I has ne e been s udied in
i o, albei se e al s uc u ally ela ed compounds ha e been epo ed as po en ially use ul
CORMs.[26–30]
In he p esen wo k, we epo spec oscopic ( luo escence spec oscopy and ci cula
dich oism) and compu a ional app oaches o cha ac e ize he in e ac ions o ALF414 wi h
bo ine se um albumin (BSA), which was chosen due o i s low cos , a ailabili y and i s 76%
sequence homology wi h HSA. This ype o undamen al in o ma ion on he in e ac ion o
ine o ganome allic coo dina ion sphe es wi h se um albumin will ce ainly p o e use ul o
he u u e design o pha macologically imp o ed CORMs.
Resul s
ALF414 (Fig 1) belongs o he amily o he so-called 4-legged piano s ool complexes. I s low
solubili y in wa e is allegedly due o he o ma ion o H-b idged dime s in he solid s a e, as
shown in he X- ay c ys al s uc u e o he complex[25]. Howe e , es s in concen a ions
50μM could be pe o med in aqueous solu ion. Abo e his alue, MeOH/H
2
O o DMSO/
Fig 1. ALF414 s uc u e.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g001
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 3 / 15
H
2
O mix u es we e used as sol en . An unusual p ope y o his ca boxylic acid is i s high pK
a
alue: 8.29±0.03 (de e mined in 1:1 H
2
O:dioxane). Fo compa ison, he pK
a
o ace ic acid in
wa e is 4.8. When dissol ed a pH >10, ALF414 decomposes ai ly quickly. I is s able in ai
o manipula ion. I is also s able o se um, as no CO is eleased a e 6 h when he compound
is incuba ed wi h pu e Fe al Bo ine Se um (FBS), which has BSA as i s main componen .
ALF414 is no e y cy o oxic, since, unde he condi ions o he MTT assay, he iabili y o
HepG2 cells ea ed wi h ALF414 (100μM in 10% MeOH/H
2
O) o 24 h is 100%, whe eas ha
o RAW 264.7 cells ea ed wi h ALF414 unde he same condi ions is 80%. The e o e, ALF414
has p ope ies compa ible wi h i s po en ial use as an o ganome allic ine CORM. The ac
ha i was ne e used o his end in i o is no ele an he e since ou ocus is on he cha ac-
e iza ion o he in e ac ions o se um albumin wi h a molecule wi h his kind o s uc u e and
ubiqui ous ligands in i s ou e sphe e. ALF414 is used he e a p o o ype o s udy he in e ac ion
o his ype o CORMs wi h se um albumin.
One impo an cha ac e is ic o ALF414 is he pola i y o i s ligand sphe e. On one hand, i
possesses he hyd ophobic ligands CO and Cp, whe eas on he o he hand, i possesses a ca -
boxylic unc ion, which can suppo pola and H-bonding in e ac ions wi h o he biological
molecules, namely p o eins. In ac , om he in e molecula in e ac ion poin -o - iew ALF414
is much close o egula o ganic d ugs han he labile, wa e soluble CORM-3 o ALF186 and
i s in e ac ions wi h anspo p o eins should be amenable o s udy h ough he classical
me hods applied o small molecule o ganic d ugs. This si ua ion is impo an in he con ex o
he op imiza ion o o ganome allic CORMs, which ha e a globula ype o s uc u e[31,32]
con as ing wi h he mo e common, ela i ely la s uc u es o mos d ugs.[31,32] To he bes
o ou knowledge, no molecula in e ac ions o his kind o CORMs wi h anspo p o eins
ha e e e been epo ed. Likewise, molecula dynamic s udies o MCCs wi h p o eins a e also
absen om he li e a u e.
Fluo escence spec oscopy
Fluo escence quenching was used o e alua e he s uc u al changes o BSA upon i s in e ac-
ion wi h ALF414. The in insic luo escence o BSA is mainly due o i s yp ophan (T p) esi-
dues loca ed in hyd ophobic ca i ies in he subdomains IIA (T p
213
) and IB (T p
134
). The
loca ion o he T p esidues is shown in Fig 2.
Fig 2. Loca ion o he yp ophan esidues on he s uc u e o BSA. Residues T p
134
and T p
213
a e ep esen ed as
g een and blue sphe es, espec i ely.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g002
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 4 / 15
In gene al, he luo escence quan um yield o T p esidues dec eases wi h i s inc eased
exposu e o he aqueous en i onmen and p oximi y o pola molecules. In ou expe imen s,
BSA ini ial luo escence in ensi y wi h emission a 340 nm (I
0
) was pa ially quenched by
ALF414. Inc eased concen a ions o ALF414 p oduced a educ ion o luo escence in ensi y,
con i ming he in e ac ion o ALF414 wi h BSA (Fig 3).
Quenching mechanisms a e usually classi ied as dynamic o s a ic quenching. They can be
dis inguished by hei di e en dependence on empe a u e and iscosi y. Dynamic quenching
depends upon di usion. As highe empe a u es esul in la ge di usion coe icien s, he
bimolecula quenching cons an s a e expec ed o inc ease wi h inc easing empe a u e. In
con as , inc eased empe a u e is likely o esul in dec eased s abili y o complexes, and hus
lowe he alues o he s a ic quenching cons an s[33]. In his con ex , we e alua ed he
quenching by ALF14 a 25 and 37 ˚C. Impo an ly, he ise o I
0
/I is also s eepe a 37 ˚C han
a 25˚C (Fig 4). The a ia ion o I
0
/I wi h he concen a ion o ALF414 is no linea and de i-
a es om he simple S e n-Volme equa ion. The e o e da a was analyzed using he Leh e
equa ion, which conside s he exis ence o a subpopula ion o he luo opho e accessible o he
quenche and ano he inaccessible o i [34] (see S1 File o de ails).
Fig 3. In insic luo escence emission spec a o BSA in he p esence o ALF414 a 25˚C and 37˚C.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g003
Fig 4. I
0
/I a ia ion wi h inc easing concen a ion o ALF414 a 25˚C and 37˚C.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g004
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 5 / 15

In o de o quan i y he quenching e icien ly, S e n-Volme cons an s (K
s
) we e ob ained
by i ing he expe imen al da a using ha equa ion (Table A in S1 File). The signi ican di e -
ence (P 0.05) be ween K
s
alue a 25˚C (0.038 ±0.003 μM
-1
) and 37˚C (0.058 ±0.001 μM
-1
)
con i ms a empe a u e-dependence on he quenching ha sugges s ha he in e ac ion
be ween BSA and ALF414 ollows a dynamic quenching a he han a s a ic mechanism. This
means ha no s able complex is o med be ween he p o ein T p esidues ( luo opho es) and
ALF414 (quenche ). Rega dless o he empe a u e,
b
alues ( ac ion o ligh emi ed by he
luo opho es accessible o he quenche (Table A in S1 File)) close o 0.5 we e ob ained (0.49
and 0.53 o 25˚C and 37˚C, espec i ely). This clea ly shows ha one o he wo BSA T p esi-
dues is accessible o he quenche , while he o he is no . Mo e impo an ly in he con ex o
he objec i es o he p esen wo k, hese da a indica e ha upon binding o BSA, ALF414 in e -
ac s wi h one (and only one) o he p o ein’s T p esidues.
Ci cula dich oism spec oscopy
Ci cula dich oism (CD) measu emen s we e used o ob ain in o ma ion abou he seconda y
s uc u e o BSA and i s changes upon binding o MCCs. Fig 5 shows he a -UV spec a o
BSA in he absence and p esence o se e al concen a ions o ALF414. BSA in solu ion is
mos ly α-helical wi h wo well-de ined ellip ici y alues a 208 and 222 nm. These CD da a
show ha he e was no al e a ion in he BSA spec a wi h inc easing concen a ions o ALF414,
indica ing ha he ligand in e ac ion does no change he seconda y s uc u e o he p o ein.
Simula ion s udies
Molecula docking was used o in es iga e he p e e ed binding mode o ALF414 o BSA and
p edic he a ini y be ween hese wo molecules. In ou speci ic case, we ound many di e en
binding si es sca e ed a ound he p o ein wi h simila ene gies (Fig A in S1 File), and, he e-
o e, hese esul s we e no conclusi e.
One o he la ges limi a ions o he molecula docking me hod used he e is he ac ha he
p o ein is conside ed igid, which can comp omise he accu acy o he p edic ions. In o de o
ci cum en his limi a ion and ake p o ein lexibili y in o accoun , we decided o use molecu-
la dynamics (MD) simula ions o unde s and he mechanism o in e ac ion be ween he wo
molecules.
Fig 5. CD spec a o ee BSA and BSA complexed wi h ALF414. The mean esidue mola ellip ici y (MRME) is
plo ed as a unc ion o he wa eleng h.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g005
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 6 / 15
MD simula ions o a sys em con aining BSA and ALF414 in explici aqueous sol en we e
pe o med, in which all he molecules we e comple ely lexible. A la ge numbe o simula ions
(50) and ligands (25 in each simula ion) we e used o ob ain s a is ically signi ican esul s.
Visual inspec ion o he ajec o ies e ealed ha he ligand molecules apidly app oach he
p o ein su ace and co e mos o i . This is no su p ising, gi en ha he ligands a e qui e
hyd ophobic a neu al pH. These molecules bind o many di e en egions, umbling on he
p o ein su ace. I is no possible o de ec an ob ious p e e ed binding si e by isual inspec-
ion o he ajec o ies. The compa ison o he s uc u al p ope ies o BSA in he p esence and
absence o ALF414 showed ha he e a e no signi ican changes on he p o ein no ha i
changes upon in e ac ing wi h he ligand (Fig C in S1 File).
To ind he p e e ed binding si es o ALF414, we analyzed he p obabili y o in e ac ion o
each esidue wi h his ligand ( esidue occupancy). As i can be obse ed in Fig 6, he e a e se -
e al esidues o BSA ha equen ly in e ac wi h ALF414, which is consis en wi h he docking
p edic ions. A sequence alignmen o bo ine and human se um albumin (Fig D in S1 File)
shows ha he egions o in e ac ion wi h ALF414 ound in he simula ions a e conse ed
be ween he wo p o eins (73% o he in e ac ing esidues a e conse ed and 10% a e no con-
se ed bu ha e simila p ope ies). This indica es ha he esul s ob ained a e meaning ul in a
human con ex whe e he CORM molecules would be anspo ed by HSA.
One o he mos popula ed binding si es con ains a T p esidue (T p
134
), which can explain
he obse ed luo escence quenching (a snapsho o a simula ion whe e ALF414 is in e ac ing
wi h T p
134
is shown in Fig 7B). This esul is also consis en wi h he docking calcula ions,
which p edic ed ha one o he p e e ed binding si es is loca ed nea T p
134
. Bo h in he dock-
ing p edic ions and in MD simula ions, only T p
134
and no T p
213
in e ac s wi h ALF414,
which is in e y good ag eemen wi h he luo escence spec oscopy da a indica ing ha only
one T p esidue is quenched.
The MD simula ion esul s also indica e ha he in e ac ion be ween T p
134
and ALF414 is
qui e dynamic and uns able (no s able complex is o med), as i can be obse ed in Mo ie_1.
Fig 6. Binding si es o ALF414 on he su ace o BSA. The esidues ha in e ac wi h he ligand o mo e han 30% o he o al simula ion ime a e
highligh ed in o ange. Residues T p
134
and T p
213
a e ep esen ed as g een and blue sphe es, espec i ely.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g006
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 7 / 15
Once again, his obse a ion is in line wi h he luo escence analysis sugges ing ha he in e ac-
ion be ween BSA and he ligand ollows a dynamic quenching, a he han a s a ic mechanism.
In o de o de e mine he ole played by each moie y o he ligand (Cp ing, ca boxyl g oup
and he CO g oups), we analyzed hei in e ac ion wi h he p o ein sepa a ely (Fig 7A). The
CO and ing moie ies in e ac much mo e equen ly wi h BSA han he ca boxyl g oup. This
shows ha he in e ac ion o his ligand wi h he p o ein is domina ed by hyd ophobic con-
ac s, which is in line wi h p e ious s udies ocusing on he in e ac ion o human se um albu-
min wi h anionic su ac an s[35]. Hyd ogen bonds be ween he ca boxyl g oup and he
p o ein also occu , al hough no e y equen ly (a snapsho o eplica e 7, whe e ALF414 is
in e ac ing wi h se e al esidues h ough hyd ophobic con ac s and hyd ogen bonding, is
shown in Fig 7B).
One o he biological oles o se um albumin is o anspo molecules, including non-es e -
i ied a y acids (FA). C ys allog aphic s udies o HSA in he p esence o FA ha e been used o
Fig 7. (A) P obabili y o in e ac ion o he h ee moie ies o ALF414 wi h BSA (B) Snapsho o eplica e 7, whe e
ALF414 is in e ac ing wi h se e al esidues, including T p
134
, h ough hyd ophobic con ac s and hyd ogen
bonding.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g007
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 8 / 15
iden i y he binding si es o hese molecules and showed ha he e a e se en FA-binding si es
dis ibu ed h oughou he p o ein[36]. The compa ison o he binding si es o ALF414 o BSA
wi h he binding si es o hexadecanoic acid o HSA (Fig 8) shows ha some o he ALF414
binding si es a e adjacen o he ones used by hexadecanoic acid, al hough only a ew esidues
in e ac wi h bo h subs a es. This is in line wi h he unspeci ic ole o se um albumin, which
can anspo di e en molecules ha end o bind he hyd ophobic pocke s o he p o ein and
in some cases also o m pola in e ac ions. This esul is also consis en wi h p e ious s udies
showing ha he p o ein uses he same binding si es o accommoda e di e en ligands,
al hough hey do no always in e ac wi h he same esidues. In ac , s uc u al s udies ha e
shown ha se e al d ugs can co-bind he in he same egion o he p o ein a he same ime
wi hou o e lapping, which in many cases c ea es a syne gis ic e ec .[37]
Since ALF414 is bulkie and mo e globula han linea -chain a y acids i does no ha e
access o some o he mos occluded binding si es used by hese molecules.
Discussion
The in e ac ion o BSA wi h he CORM p o o ype ALF414, which belongs o he amily o he
so-called 4-legged piano s ool complexes, was analyzed o he i s ime using a combina ion
o spec oscopic and molecula simula ion me hods.
The analysis o he luo escence emission spec a o BSA in he p esence o inc easing con-
cen a ions o ALF414 e ealed ha he ini ial luo escence in ensi y o he p o ein was pa -
ially quenched by he ligand, which indica es ha ALF414 in e ac s wi h a leas one o he
T p esidues o BSA, hus educing he p o ein’s in insic luo escence. This analysis u he
showed ha he ligand in e ac s wi h only one o he wo T p esidues o BSA, since he ac-
ion o ligh emi ed by he luo opho es accessible o he quenche was close o 0.5. Mo eo e ,
he empe a u e dependence o he quenching by ALF414 showed ha i ollows a dynamic
mechanism, which indica es ha he BSA-ALF414 in e ac ion is dynamic. One could e en u-
ally eason ha he luo escence quenching, ins ead o being caused by a di ec in e ac ion
wi h ALF414, could al e na i ely be due o a majo con o ma ional change o he p o ein
induced by ALF414, leading o a comple e change o he mic oen i onmen o one o he T p
esidues. Howe e , his hypo hesis is uled ou by he ci cula dich oism and molecula
dynamics da a, which indica es ha he e a e no majo changes in he BSA s uc u e in he
absence and p esence o ALF414.
Fig 8. Compa ison o he binding si es o ALF414 o BSA wi h he binding si es o hexadecanoic acid o HSA. The
esidues ha in e ac wi h ALF414 in his wo k a e colo ed in o ange, he esidues ha bind hexadecanoic acid
molecules a e colo ed in pu ple and he esidues ha in e ac wi h bo h ligands a e colo ed in ed.
h ps://doi.o g/10.1371/jou nal.pone.0204624.g008
In e ac ions o bo ine se um albumin wi h a molybdenum(II) ca bonyl complex
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204624 Sep embe 27, 2018 9 / 15