1
Reduced s uc u al igidi y o MDMX p o ein
enhances binding o TP53 mRNA
Ma ina Kuce iko a1,2, Ond ej Bonczek1, Vanesa Oli a es-Illana3, And es Rod iguez-
Rod iguez3, Jose G. Samped o3, Lenka He nycho a1, Vacla H abal1,4, Pa lina
Za loukalo a1, Rado an K ejci 1, Robin Fah aeus1,5,6, Philip J. Coa es1, Bo i oj Voj esek1,7*,
Lucia Ma inko a1*
1 RECAMO, Masa yk Memo ial Cance Ins i u e, 602 00, B no, Czech Republic
2 Na ional Cen e o Biomolecula Resea ch, Facul y o Science, Masa yk Uni e si y, 625
00, B no, Czech Republic
3 Ins i u o de Fisica, Uni e sidad Au onoma de San Luis Po osi, 78290 San Luis Po osi,
Mexico
4 Depa men o Expe imen al Biology, Facul y o Science, Masa yk Uni e si y, 625 00, B no,
Czech Republic
5 Inse m UMRS1131, Ins i u de Gene ique Moleculai e, Uni e si e Pa is Ci e, Hopi al S .
Louis, 75010, Pa is, F ance
6 Depa men o Medical Biosciences, Umea Uni e si y, 901 87, Umea, Sweden
7 Labo a o y o G ow h Regula o s, Ins i u e o Expe imen al Bo any, The Czech Academy o
Sciences, 779 00, Olomouc, Czech Republic
Co espondence: lucia.ma inko [email protected], bo i oj. oj [email protected]
Abs ac
The wo mu ine double minu e (MDM) amily membe s, MDM2 and MDMX a e a
well-es ablished nega i e egula o o p53 ac i i y. Unde DNA damage condi ions, MDM2
and MDMX a e phospho yla ed nea hei RING domains (se ine 395 a MDM2 and se ine
403 a MDMX) and swi ch o ac as p53 posi i e egula o s. MDMX binds o TP53 mRNA
and ac s as a chape one o RNA s uc u e, enabling MDM2 o bind. This in e ac ion
enhances TP53 mRNA ansla ion, leading o inc eased p53 p o ein p oduc ion. While he
biological signi icance o his in e ac ion has been desc ibed, he speci ic ea u es o he
MDMX-RNA in e ac ion emain poo ly unde s ood. We used a ious MDMX p o ein
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cons uc s o cha ac e ize binding o TP53 mRNA and iden i ied ha he in e ac ion media ed
by he RING domain is modula ed by he p esence o o he domains. Hyd ogen-deu e ium
exchange mass spec ome y (HDX-MS) and binding assays in high sal condi ions and
a ious pH demons a e ha he whole p o ein pa icipa es in RNA in e ac ion, wi h he C-
e minal domain likely p o iding he con ac wi h RNA by elec os a ic o ces. We show ha
p o ein s uc u al changes induced by he chela ing agen EDTA o he educing agen TCEP
enhances RNA binding by p omo ing pa ial s uc u al des abiliza ion o he p o ein. Ou
indings sugges ha he MDMX/TP53 mRNA in e ac ion is complex, wi h he RING domain
binding o RNA and being suppo ed by he en i e p o ein, which ac s as a sca old o he
RNA in e ac ion. These esul s con ibu e o a be e unde s anding o MDMX´s ole in TP53
mRNA binding and p o ide aluable insigh s o u u e in es iga ion o he MDM2-MDMX-
TP53 mRNA complex, which is c ucial o p53 s abiliza ion and ac i a ion unde DNA-
damaging condi ions.
Keywo ds: MDMX-RNA in e ac ion; TP53 mRNA; RING domain; HDX-MS mapping
In oduc ion
MDMX is an oncop o ein ha is ampli ied and o e exp essed in a ious cance s,
leading o diminished p53 ac i i y and p omo ing umou g ow h and su i al [1,2]. This
abe an exp ession is o en mu ually exclusi e wi h TP53 mu a ions and MDM2
ampli ica ion, and is equen ly associa ed wi h poo p ognosis in cance pa ien s,
highligh ing i s c ucial ole in umo igenesis [1,2]. Unde no mal condi ions, MDMX, in
collabo a ion wi h i s homolog MDM2, plays a key ole in main aining low le els o p53
p o ein by di ec ing p53 o p o easomal deg ada ion [1]. MDMX also exe s p53-
independen unc ions, including egula ion o cell cycle, genomic ins abili y, and me abolism
[3–5]. Du ing DNA damage, a phospho yla ion cascade igge ed by a axia- elangiec asia
mu a ed (ATM) kinase dis up s he p53-MDM2-MDMX in e ac ion, esul ing in inc eased p53
p o ein le els and ac i a ion [6]. ATM di ec ly phospho yla es MDM2 a se ine 395 and
MDMX a se ine 403. As a esul , MDMX and MDM2 shi hei unc ion o ac as posi i e
egula o s o p53 ac i i y by di ec ly binding TP53 mRNA and enhancing i s ansla ion [7]. In
his con ex , MDMX ac s as an RNA chape one p o ein o TP53 mRNA and enables he
e icien binding o MDM2 [8]. Recen ly, i was shown ha he ATM kinase also binds TP53
mRNA unde s ess condi ions, and MDMX p e en s his in e ac ion and subsequen
in e ac ion wi h Nijmegen b eakage synd ome 1 (NBS1), a componen o he MRN (MRE11–
RAD50–NBS1) complex in ol ed in DNA damage ecogni ion and epai [9].
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S uc u ally, MDMX is an in insically diso de ed p o ein wi h se e al o ganized
domains, including he p53-binding domain a i s N e minus, he WWW elemen , he acidic
domain, he zinc- inge domain, and he RING domain (Figu e 1A). The RING domain,
loca ed a he C- e minus, is c i ical o he e odime iza ion wi h MDM2 [10]. Recen in cellulo
s udies ha e also desc ibed ha homodime iza ion o MDMX in luences i s binding o p53
[11]. The RNA-binding ac i i y o MDMX has been mapped o i s RING domain [8]. Due o i s
s uc u al o ganiza ion, MDMX exhibi s signi ican lexibili y [12]. The e o e, s udies o
domain unc ions, complemen ed by ull-leng h p o ein analysis, can p o ide a mo e
comp ehensi e unde s anding.
We cha ac e ized key aspec s o he MDMX-RNA in e ac ion, p o iding new insigh s
in o how MDMX modula es TP53 mRNA and i s po en ial oles. We con i med ha he RING
domain is essen ial o TP53 mRNA in e ac ion, while o he domains play modula o y oles
likely due o s uc u al o ganiza ion. Binding a a ious pH and high sal condi ions, oge he
wi h HDX-MS, e ealed ha p o ein-RNA binding is pa ially d i en by elec os a ic o ces a
he p o ein's C- e minus, bu he en i e p o ein pa icipa es in binding and s abilizes he
in e ac ion. Fu he mo e, we in es iga ed how educing TCEP and chela ing condi ions
EDTA a ec bo h he s uc u al o ganiza ion o MDMX and i s TP53 mRNA-binding capaci y,
wi h con o ma ional des abiliza ion enhancing RNA in e ac ion. The ole o MDMX as an
RNA-binding p o ein adds an impo an dimension o i s egula o y epe oi e. A deepe
unde s anding o hese in e ac ions could lead o no el he apeu ic app oaches in cance
ea men .
Ma e ial and me hods
Cloning and pu i ica ion o p o ein cons uc s
The o iginal pu i ica ion p o ocol [12] was modi ied as ollows: cDNA o MDMX wild- ype (WT)
o S403D cons uc s we e cloned in o HIS- agged ec o s pET28B (Me ck Millipo e,
Da ms ad , Ge many) and pDEST17 (The mo Fishe Scien i ic, Wal ham, MA, USA). The
Se 403- o-Asp mu a ion mimics ATM kinase–media ed phospho yla ion a his si e in
esponse o DNA damage. The insoluble p o ein ac ion was esuspended in guanidine
bu e (6 M guanidine hyd ochlo ide, 50 mM T is (pH 8.0), 150 mM NaCl, 10 mM imidazole,
2 mM MgCl₂) and incuba ed a 37 °C o 90 min, wi h Tu bonuclease om Se a ia
ma cescens (Me ck Millipo e) added du ing he inal 15 min o incuba ion o emo e esidual
nucleic acids. The Ni-NTA Aga ose (Qiagen, Hilden, Ge many) was washed wi h 0.1 M T is
(pH 8), 2 M NaB , 0.5 M NaCl, and 10 mM 2-me cap oe hanol. P o eins we e elu ed om he
washed esin using 5 ml (5 ac ions o 1 ml) o elu ion bu e (50 mM T is (pH 8), 300 mM
Imidazole, 10 µM ZnSO4·7H2O). Elu ed p o eins we e dialyzed o e nigh a 4 °C in 50 mM
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T is (pH 8), 150 mM NaCl, and 10 µM ZnSO₄. A e dialysis, p o eins we e concen a ed
using 10 kDa MWCO Amicon Ul a-4 cen i ugal il e s (Me ck Millipo e).
Sodium dodecyl sulpha e-polyac ylamide gel elec opho esis (SDS-PAGE)
SDS-PAGE was pe o med as desc ibed p e iously [13]. 300 ng o pu i ied p o ein was
loaded pe lane o elec opho e ic sepa a ion, and gels we e s ained wi h Coomassie
b illian blue o 30 min. P o ein isualiza ion was pe o med using ChemiDoc Imaging (Bio-
Rad, He cules, CA, USA).
Elec opho e ic mobili y shi assay (EMSA)
In i o ansc ip ion o TP53 mRNA was pe o med using he mMESSAGE mMACHINE™
T7 T ansc ip ion Ki (The mo Fishe Scien i ic) acco ding o he manu ac u e ’s ins uc ions.
The linea ized pcDNA3-p53 plasmid (encoding nucleo ides 1–240) [8] was used as he DNA
empla e. A o al o 100 ng o RNA was mixed wi h pu i ied p o ein (in a 10 µL eac ion) a
RNA:p o ein mola a ios speci ied in he igu es, using binding bu e (150 mM NaCl, 50 mM
T is, 1 mM TCEP, 10 µM ZnSO₄, pH 7.5) along wi h addi ional condi ions ou lined in he
igu es. Samples we e incuba ed a oom empe a u e o 20 min be o e being loaded on o
1.2% aga ose gels. The gels we e pos -s ained wi h GelRed nucleic acid s ain (Bio ium,
F emon , CA, USA) o 40 min. Fo p o ein de ec ion ollowing he GelRed s aining, he same
gels we e ixed in 50% me hanol and 7% ace ic acid o 20 min, and subsequen ly s ained
o e nigh wi h SYPRO™ Ruby p o ein gel s ain (The mo Fishe Scien i ic). Bo h gels we e
imaged using ChemiDoc Imaging (Bio-Rad). Densi ome ic analysis was pe o med o all
EMSA gels, and he in ensi y o ee RNA was no malized o he RNA-only con ol. Da a
ep esen mean ± SEM om h ee independen biological eplica es (n = 3). S a is ical
signi icance was de e mined using wo- ailed S uden ’s - es , wi h he ollowing anno a ions:
*p < 0.05, **p < 0.01, ***p < 0.001. In addi ion, some compa isons be ween speci ic
condi ions a e indica ed in he igu es using al e na i e symbols: •p < 0.05, ••p < 0.01, •••p <
0.001.
HDX-MS
HDX-MS o he HDMX-S403D p o ein was conduc ed in bo h ligand- ee and ligand-bound
con o ma ions wi h TP53 mRNA, ollowing he expe imen al p ocedu es desc ibed in [14,15].
De ailed me hodology and HDX-MS da a a e a ailable in he P o eomeXchange (PX)
Conso ium [16] ia he P o eomics Iden i ica ions (PRIDE) [17] eposi o y (Da ase iden i ie :
PXD060074; Use name: e iewe _px[email p o ec ed]; Passwo d: LyazwVQgkNEC).
Ci cula dich oism (CD) spec oscopy
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Fa -UV CD spec a o MDMX-S403D FL and S403D (322-490) we e eco ded using a Jasco
J-1500 Spec ome e (Jasco Inc., Eas on, MD, USA) in a wa eleng h ange o 180 o 280 nm.
Measu emen s we e pe o med a a p o ein concen a ion o 0.5 mg/ml in phospha e bu e
(pH 7.5), using qua z cu e es wi h a 0.1 cm pa h leng h.
In insic luo escence
The emission luo escence spec a o he p o eins (a 0.5 mg/ml concen a ion) in phospha e
bu e (pH 7.5) o binding bu e we e eco ded using an Agilen Technologies Ca y Eclipse
Fluo escence Spec opho ome e (Agilen Technologies, San a Cla a, CA, USA).
Measu emen s we e pe o med a oom empe a u e in qua z cu e es wi h a 1 cm pa h
leng h. The exci a ion wa eleng h was se o 280 nm, and blank measu emen s (wi hou
p o ein) we e eco ded and sub ac ed om he expe imen al spec a.
Resul s
Domain-speci ic con ibu ions o he RNA-binding ac i i y o he p o ein
To cha ac e ize he RNA-binding p ope ies o MDMX, we p epa ed ecombinan p o eins in
bo h he wild- ype (WT) o m and a phosphomime ic mu a ion a se ine 403 (S403D), which
enhances a ini y o TP53 mRNA [8]. Fo he S403D mu an , we gene a ed ou MDMX
a ian s: ull-leng h (FL; 1–490), a unca ed cons uc lacking he N- e minal domain (128–
490), a cons uc lacking he C- e minal egion including he RING domain (1–436), and a C-
e minal agmen con aining only he RING domain (322–490). In addi ion, wo WT
cons uc s we e p oduced: ull-leng h (FL; 1–490) and he RING-con aining C- e minal
a ian (322–490) (Figu e 1A). All ecombinan p o eins we e analysed using EMSA. The
TP53 mRNA agmen (1–240), co esponding o he i s 240 nucleo ides o he ull-leng h
p53 coding sequence, was syn hesized by in i o ansc ip ion. This egion includes he
in e nal ibosome en y si e (IRES) o he p53/47 iso o m and has p e iously been iden i ied
as he MDMX-in e ac ing egion [8]. Binding assays we e pe o med using 100 ng o TP53
mRNA (nucleo ides 1–240), he ea e e e ed o as “RNA”, and inc easing concen a ions o
ecombinan MDMX p o eins. The RNA-p o ein o ma ion was indica ed as a educ ion o
ee RNA and o p o ein a ian s FL (Figu e 1B and 1F) and unca ion 128-490 (Figu e 1C)
as a shi ed gel band. The C- e minal cons uc s 322-490 (Figu e 1E) mig a ed poo ly in o
gel, o en appea ing as a smea o emaining apped in he gel wells. This beha io is likely
due o he highe pI and s uc u al p ope ies o he cons uc , al hough he possibili y o
agg ega ion canno be en i ely uled ou . Mo eo e , p o ein con o ma ional changes may
educe RNA accessibili y in complex, he eby hinde ing i s de ec abili y by s aining unde
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ce ain condi ions. The e o e, we e alua ed he educ ion o ee RNA a he han he signal
in ensi y o he p o ein–RNA complex o compa e he RNA-binding a ini y o all p o ein
a ian s (Figu e 1H), as well as p o ein-RNA a ini y unde o he condi ions (shown in la e
igu es). As expec ed, in he absence o p o ein (lane 1:0), only ee RNA was de ec ed,
se ing as a e e ence o he unbound RNA signal (Figu e 1B-G). MDMX-S403D FL (Figu e
1B) showed enhanced binding o TP53 mRNA, as e idenced by a educ ion in ee RNA and
he appea ance o a shi ed RNA–p o ein complex a a 1:25 RNA:p o ein mola a io, wi h
nea -comple e o ma ion o he complex obse ed a 1:100. The S403D a ian lacking he
N- e minal domain, MDMX-S403D (128–490), exhibi ed educed RNA binding (Figu e 1C),
sugges ing ha he N- e minal egion modula es o acili a es he RNA in e ac ion. The
MDMX-S403D (1–436) a ian , which lacks he RING domain, did no bind TP53 mRNA a
he es ed concen a ions (Figu e 1D), indica ing ha he RING domain is essen ial o RNA
in e ac ion. In con as , he C- e minal a ian MDMX-S403D (322–490), con aining he
RING domain, educed ee RNA ac oss all es ed RNA:p o ein a ios, indica ing highe
a ini y han ull-leng h MDMX-S403D (Figu e 1E), as p e iously shown by RNA-ELISA [8].
Howe e , smea a he han disc e e bands a e isible as complexes, lea ing open he
possibili y ha p o ein agg ega ion con ibu es o he obse ed binding. We also compa ed
RNA binding o he wo WT o ms o MDMX, ull-leng h (1–490) (Figu e 1F) and he RING-
con aining C- e minal a ian (322–490) (Figu e 1G), and con i med p e ious indings [8] ha
WT p o eins bind TP53 mRNA less e icien ly han hei phosphomime ic coun e pa s
(Figu e 1B and 1E). Al hough po en ial agg ega ion o he C- e minal a ian cons uc s may
con ibu e o he obse ed binding pa e n, he indings none heless sugges ha he RING
domain plays an impo an ole in he MDMX–TP53 mRNA in e ac ion. The S403D
phosphomime ic mu a ion enhances binding a ini y, whe eas o he domains po en ially
mask o modula e he RNA-binding in e ace.
Mapping he RNA in e ac ion si e o he MDMX p o ein
MDMX is a highly lexible p o ein due o diso de ed egions be ween i s s uc u ed domains
[12] implying ha ce ain p o ein o RNA in e ac ions may exe allos e ic e ec s ha
egula e i s in e ac ome [18]. To iden i y he RNA-binding si e on MDMX-S403D FL (1-490)
and o s udy i s allos e ic e ec , we pe o med HDX-MS a h ee ime in e als: 10 s (Figu e
S1A), 120 s (Figu e 2A), and 1 800 s (Figu e S1B). By his me hod, di ec nucleic acid -
p o ein in e ac ions a e seen as deu e a ion supp ession, and ea angemen s o p o ein
s uc u e due o ligand binding a e seen as inc eased deu e a ion o s uc u e opening o
dec eased deu e a ion o s uc u e closing. Ou esul s showed no speci ic RNA-binding si e
wi hin he RING domain, bu small deu e a ion changes we e obse ed h oughou he en i e
p o ein, wi h he mos no iceable opening in he N- e minal domain (100-120 aa) (Figu e 2A),
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suppo ing he no ion ha he en i e p o ein pa icipa es, ei he di ec ly o indi ec ly, in RNA
binding. The RING domain (440–490) exhibi s low deu e a ion (Figu e 2A), sugges ing
masking by o he p o ein domains. The inabili y o iden i y a di ec RNA con ac si e may
e lec limi a ions o he expe imen al se ings, including es ic ed accessibili y o he RING
domain unde he assay condi ions, he po en ial absence o a well-de ined binding in e ace,
o he inhe en ins abili y, ansien na u e, and possible non-speci ici y o he in e ac ion, all
o which could p e en eliable de ec ion.
We used inc easing sal concen a ions in he binding assay o elucida e he na u e o he
in e ac ion be ween MDMX and RNA. The C- e minal cons uc MDMX-S403D (322–490)
educed nea ly all a ailable RNA a 150 mM NaCl (Figu e 2B, lane 2, quan i ied in Figu e 2D)
occu ing along wi h he smea on he gel ha sugges s RNA-p o ein complex o ma ion.
Howe e , as he sal concen a ion inc eased (s a ing a 250 mM NaCl), he amoun o ee
RNA also inc eased, sugges ing ha highe sal concen a ion dis up ed elec os a ic
in e ac ions equi ed o RNA–p o ein complex o ma ion (Figu e 2B, lanes 4, 6 and 8). In
con as , he RNA–p o ein complexes o med by MDMX-S403D FL we e esilien o
inc easing sal concen a ions (150–600 mM), emaining s able e en a highe ionic s eng h
(Figu e 2C, quan i ied in Figu e 2D). This sugges s ha he in e ac ion is suppo ed by
addi ional, non-elec os a ic o ces. Since sal concen a ion may in luence he binding
e iciency o GelRed s ain o RNA (Figu e S2), each EMSA gel included ee RNA con ols
loaded a he co esponding NaCl concen a ion. Al oge he , hese indings sugges ha he
C- e minal cons uc domain binds RNA ia elec os a ic o ces ha a e no de ec able by
HDX-MS, while o he MDMX domains may pa ially shield he RING in e ace and con ibu e
o s abiliza ion o he RNA-p o ein complex.
The e ec o pH on he binding o MDMX-S403D o RNA
To e alua e u he he elec os a ic con ibu ion o p o ein-RNA in e ac ion, we examined
how he a ia ions in pH (7.0, 7.5, and 8.0) will in luence he RNA binding. The MDMX-
S403D FL p o ein has an isoelec ic poin (pI) o 5.1, whe eas he C- e minal cons uc
MDMX-S403D (322–490) has a pI o 7.6. The pH shi om 7.0 o 8.0 p ima ily a ec s
cys eine and his idine esidues loca ed mainly in he C- e minus egion o MDMX, al e ing
hei p o ona ion s a e and esul ing in a mo e nega i e o e all cha ge (Figu e S3) [19,20].
Fo he MDMX-S403D FL cons uc (Figu e 3A, quan i ied in 3C), no signi ican di e ence in
RNA binding was obse ed ac oss he es ed pH ange (Figu e 3A, lanes 2-4, uppe panel).
RNA alone exhibi ed a consis en mig a ion pa e n a pH 7.5 (lane 1) and pH 8.0 (lane 8).
The MDMX-S403D (322–490) cons uc (Figu e 3B, quan i ied in 3C) showed small bu
consis en educ ion in RNA binding as pH inc eased (Figu e 3B, lanes 2-4, uppe panel),
co esponding o educed p o ona ion a highe pH alues leading o inc eased nega i e
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cha ge and weakened elec os a ic in e ac ions. To de e mine whe he al e ed p o ein
mig a ion con ibu ed o he obse ed e ec s, we s ained he aga ose gels wi h SYPRO
Ruby (Figu e 3A and 3B, lowe panels). Ac oss all es ed pH condi ions, he mig a ion o
bo h cons uc s emained unchanged, whe he in ee o m (lanes 5–7) o as pa o an
RNA–p o ein complex (lanes 2–4), indica ing ha di e ences in EMSA esul s a e no due o
al e ed p o ein mobili y. Mo eo e , hese esul s show ha he C- e minal unca ion (322–
490) alone pene a es poo ly in o he gel wells (Figu e 3B, lowe panel, lanes 5-7) and is
only d awn e icien ly in o he gel in he p esence o RNA (lane 2-4), appea ing as a smea ,
whe eas he ull-leng h p o ein pene a es he gel mo e e icien ly and o ms disc e e
complexes isible as a band (Figu e 3A, lowe panel). These esul s e lec he elec os a ic
na u e o RNA binding by he C- e minal egion and highligh he ole o he en i e p o ein in
s abilizing he RNA in e ac ion ac oss a b oade pH ange.
Condi ions in luencing accessibili y o he MDMX RING domain egula e RNA binding
We hypo hesized ha MDMX binds RNA ia i s RING domain, wi h o he domains po en ially
masking he in e ac ion in e ace. To cha ac e ize he s uc u al ea u es wi hin he RING
domain ha a e c i ical o RNA in e ac ion, we examined he ole o zinc coo dina ion and
disul ide bond in eg i y in MDMX-S403D FL binding. The RING domain o MDMX consis s o
a C2H2C4 s uc u e wi h wo cys eines (C1 and C2), wo his idines (H3 and H4), and ou
cys eines (C5–C8), which coo dina e wo Zn²⁺ions essen ial o s abilizing i s olded s a e
[21,22]. Mo eo e , MDMX con ains an addi ional Zn- inge domain spanning esidues 290–
322. To in es iga e he ole o zinc ions and hei chela ion by EDTA in RNA binding, we
pe o med EMSA using he MDMX-S403D FL p o ein (Figu e 4A, quan i ied in 4B) and he
C- e minal a ian MDMX-S403D (322–490) (Figu e S4A, quan i ied in S4B) in he p esence
o zinc concen a ions p omo ing p ope p o ein olding (10 µM Zn²⁺), an excess o ee Zn²⁺
(100 µM), and 1 mM EDTA combined wi h a ying zinc concen a ions. Fo MDMX-S403D
FL, excess zinc ions e ealed dec ease in RNA binding seen as ee RNA inc ease (Figu e
4A, uppe panel, lane 3). In con as , he addi ion o EDTA enhanced RNA binding
signi ican ly, sugges ing ha zinc chela ion by EDTA acili a es in e ac ion wi h RNA (Figu e
4A, uppe panel, lane 4 and 5, quan i ied in Figu e 4B). Simila ends in RNA changes we e
obse ed o he MDMX-S403D (322–490) a ian (Figu e S4A and S4B), and upon EDTA
addi ion, his was accompanied by disappea ance o he smea ha had indica ed RNA–
p o ein complex o ma ion (Figu e S4A, uppe panel, lines 4 a 5). I has been p oposed ha
EDTA may induce un olding and po en ial agg ega ion o RING inge domains [23]. To
examine his e ec , we used SYPRO Ruby s aining o e alua e p o ein mobili y in aga ose
gels, in bo h RNA-bound and RNA- ee o ms. MDMX-S403D FL o med wo bands e en in
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he absence o RNA (Figu e 4A, lowe panel, lanes 6-8). No signi ican change in mobili y
was obse ed upon Zn²⁺ ea men (lanes 3 and 7), indica ing no majo s uc u al changes
occu ed. Howe e , in he p esence o 1 mM EDTA, we obse ed educ ion o he lowe
p o ein band in bo h RNA-bound (lanes 4 and 5) and RNA- ee o ms (lane 8), sugges ing a
s uc u al al e a ion o he p o ein. By o e laying GelRed and SYPRO Ruby s aining, we
iden i ied he uppe band as he RNA-binding o m (Figu e S4E), consis en wi h EDTA-
induced s uc u al ea angemen ha p omo es RNA in e ac ion. The C- e minal a ian
MDMX-S403D (322–490) showed no mobili y changes in esponse o zinc ions (Figu e S4A,
lowe panel, lanes 2, 3, 6 and 7). Howe e , addi ion o 1 mM EDTA induced accumula ion o
he p o ein in he gel wells accompanied by he disappea ance o he smea , sugges ing
p o ein agg ega ion (Figu e S4A, lowe panel, lanes 4, 5, and 8). Toge he , hese esul s
indica e ha zinc ions in e e e wi h RNA binding, while EDTA enhances binding,
p esumably by chela ing zinc and unmasking he in e ace. Howe e , EDTA p obably
weakens he s uc u al s abili y o he p o ein, pa icula ly o he C- e minal domain. These
indings highligh he delica e balance be ween s uc u al in eg i y and accessibili y in he
egula ion o RNA binding by he MDMX RING domain.
The MDMX p o ein con ains cys eine esidues, p ima ily loca ed in he zinc inge and RING
domains, which can o m disul ide bonds in ol ed in p o ein olding and oligome iza ion.
Howe e , unde educing condi ions p e alen in he cellula en i onmen , disul ide bonds
become uns able. To in es iga e he ole o disul ide bonds in RNA binding, we used
inc easing concen a ion o TCEP, a po en educing agen , and analysed RNA-binding
capaci y using EMSA (Figu e 4C and quan i ied in 4D). RNA–p o ein complex o ma ion was
obse ed unde na i e condi ions wi hou TCEP (lane 2) and inc eased upon ea men wi h
1 mM TCEP (lane 3). Howe e , a 5 mM TCEP, complex o ma ion by MDMX-S403D FL was
dis up ed, indica ed by he eappea ance o ee RNA (lane 4). SYPRO Ruby con i med ha
1 mM TCEP did no al e he mig a ion o MDMX-S403D FL (Figu e 4C, lowe panel, lane 3),
whe eas 5 mM TCEP p obably caused agg ega ion, wi h p o ein e ained in he well (lane 4).
Simila agg ega ion was obse ed o he RNA- ee p o ein a 5 mM TCEP (lane 7). Fo
MDMX-S403D (322–490) (Figu e S4C and quan i ied in S4D), we obse e dec ease in ee
RNA upon 1mM and 5 mM TCEP addi ion. Howe e , he SYPRO s aining e eals (Figu e
S4C, lowe panel) ha 1 mM TCEP enhanced gel pene a ion o bo h RNA-bound and RNA-
ee o ms (lanes 3 and 6), indica ing disul ide bond dis up ion. In con as , 5 mM TCEP
again led o p o ein e en ion in he well sugges ing agg ega ion (lanes 4 and 7). These
esul s sugges ha excessi e disul ide bond o ma ion can pa ially obs uc RNA binding,
whe eas hei comple e absence dis up s p o ein in eg i y. In summa y, disul ide bonds a e
essen ial o bo h he RNA-binding abili y and he s uc u al s abili y o MDMX.
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Figu e 1: TP53 mRNA binding o phosphomime ic and wild- ype MDMX p o ein
cons uc s.
(A) Schema ic ep esen a ion o MDMX p o ein cons uc s ca ying a phosphomime ic
mu a ion a se ine 403 (MDMX-S403D), shown in ou a ian s: ull-leng h MDMX-S403D FL
(1–490), a unca ed o m lacking he N- e minal domain MDMX-S403D (128–490), a
cons uc lacking he C- e minal egion including he RING domain MDMX-S403D (1–436),
and a C- e minal agmen con aining only he RING domain MDMX-S403D (322–490). The
domain a chi ec u e includes he p53-binding domain (p53BD, pink), WWW domain (blue),
acidic domain (AD, g een), zinc-binding domain (ZD, ed), and he RING domain (yellow).
(B–E) EMSA analysis o TP53 mRNA binding o he ou MDMX-S403D a ian s shown in
(A). (F, G) EMSA analysis o TP53 mRNA binding o wild- ype MDMX cons uc s: (F) ull-
leng h MDMX WT FL (1–491) and (G) he C- e minal RING-con aining agmen MDMX WT
(322–490). All ecombinan p o eins we e incuba ed wi h in i o ansc ibed TP53 mRNA
(nucleo ides 1–240) a RNA:p o ein mola a ios o 1:25, 1:50, and 1:100. F ee RNA ( a io
1:0) se ed as con ol. Reac ions we e pe o med in binding bu e (150 mM NaCl, 50 mM
T is, 1 mM TCEP, 10 µM ZnSO₄, pH 7.5). RNA–p o ein complexes and ee RNA we e
isualized on aga ose gels by GelRed s aining. B acke s indica e smea s co esponding o
p o ein–TP53 mRNA complexes. (H) Quan i ica ion o ee RNA band in ensi y om EMSA
gels shown in (B–G). Fo each RNA:p o ein a io (1:25, 1:50, 1:100), ee RNA in ensi y was
no malized o he RNA-only con ol (1:0). Colo ed cu es ep esen indi idual cons uc s: (B)
MDMX-S403D FL (1–490), ed; (C) MDMX-S403D (128–490), blue; (D) MDMX-S403D (1–
436), g een; (E) MDMX-S403D (322–490), pu ple; (F) MDMX WT FL (1–490), black; (G)
MDMX WT (322–490), g ey. Da a ep esen mean ± SEM (n = 3 biological eplica es).
Figu e 2: Cha ac e iza ion o he MDMX-S403D in e ac ion wi h TP53 mRNA by HDX-
MS and EMSA unde a ying sal concen a ions.
(A) HDX-MS analysis o ull-leng h MDMX ca ying a phosphomime ic mu a ion a se ine 403
(MDMX-S403D FL) in complex wi h TP53 mRNA. Rela i e ac ional up ake ac oss amino
acid esidues is shown o ee MDMX-S403D FL (o ange) and he MDMX-S403D FL–TP53
mRNA complex (blue) a e 120 s deu e ium exposu e. Non-exchangeable p oline esidues
a e indica ed as gaps in he p o ile. (B, C) EMSA analysis o TP53 mRNA binding o (B)
MDMX-S403D (322–490) and (C) MDMX-S403 FL unde inc easing NaCl concen a ions
(150, 250, 400, and 600 mM). P o ein–RNA complexes (lanes 2, 4, 6, 8) and co esponding
ee RNA con ols (lanes 1, 3, 5, 7) we e isualized on aga ose gels by GelRed a an
RNA:p o ein mola a io o 1:50. B acke s indica e smea s co esponding o p o ein–TP53
mRNA complexes. (D) Quan i ica ion o ee RNA band in ensi y de i ed om he EMSA gels
in (B) and (C). Da a o MDMX-S403D FL a e shown as emp y columns ( om gel C) and o
MDMX-S403D (322–490) as do ed columns ( om gel B), no malized o hei espec i e
RNA-only con ols. Da a ep esen mean ± SEM (n = 3 biological eplica es; ***p < 0.001).
Addi ional s a is ical compa isons a e shown in he igu e and al e na i ely indica ed as •p <
0.05 and •••p < 0.001.
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19
Figu e 3: E ec o pH on TP53 mRNA-MDMX in e ac ion.
(A, B) EMSA showing he e ec o pH (7.0, 7.5, and 8.0) on TP53 mRNA binding o (A)
MDMX-S403D FL and (B) MDMX-S403D (322–490). P o eins we e incuba ed wi h TP53
mRNA (lanes 2–4) o wi hou RNA (lanes 5–7) in binding bu e , adjus ed o he indica ed pH
alues. F ee RNA con ols we e included a pH 7.5 and 8.0 (lanes 1 and 8). The RNA:p o ein
mola a io was 1:50 in all eac ions. B acke s indica e smea s co esponding o p o ein–
TP53 mRNA complexes. RNA and RNA–p o ein complexes we e isualized on GelRed-
s ained aga ose gels (uppe panels), and o al p o ein was de ec ed using SYPRO Ruby
s aining o he same gels (lowe panels). (C) Quan i ica ion o ee RNA band in ensi y
de i ed om he EMSA gels in (A) and (B). Da a o MDMX-S403 FL a e shown as whi e
columns ( om gel A) and o MDMX-S403D (322–490) as g ey columns ( om gel B),
no malized o hei espec i e RNA-only con ol. Da a ep esen mean ± SEM (n = 3
biological eplica es; *p < 0.05, ***p < 0.001). Addi ional s a is ical compa isons a e shown in
he igu e and al e na i ely indica ed as •p < 0.05.
Figu e 4: Modula ion o TP53 mRNA–MDMX-S403D FL in e ac ion by zinc, EDTA, and
educing condi ions.
(A) EMSA analysis o TP53 mRNA binding o MDMX-S403D FL cons uc in he p esence o
Zn²⁺and EDTA. P o eins we e incuba ed wi h TP53 mRNA (lanes 2–5) o wi hou RNA
(lanes 6–8) in binding bu e (150 mM NaCl, 50 mM T is, 1 mM TCEP, pH 7.5) and ei he
10 µM o 100 µM ZnSO₄, in he p esence o EDTA (do ed columns) o absence o EDTA
(emp y columns). F ee RNA (lane 1) se ed as a con ol. (B, D) Quan i ica ion o ee RNA
band in ensi y om gels shown in (A) and (C), espec i ely, no malized o hei
co esponding RNA-only con ol. Da a ep esen mean ± SEM (n = 3 biological eplica es;
***p < 0.001). Addi ional s a is ical compa isons a e shown in he igu e and al e na i ely
indica ed as •p < 0.05 and ••p < 0.01. (C) EMSA showing he e ec o he educing agen
TCEP on TP53 mRNA o MDMX-S403D FL cons uc . P o eins we e incuba ed wi h TP53
mRNA (lanes 2–4) o wi hou RNA (lanes 5–7) in binding bu e con aining 0, 5, o 10 mM
TCEP. F ee RNA (lane 1) se ed as a con ol. In panels (A) and (C), RNA–p o ein
complexes we e isualized by GelRed s aining (uppe panels), and o al p o ein was
de ec ed using SYPRO Ruby s aining o he same gels (lowe panels). The RNA:p o ein
mola a io was 1:50 in all eac ions.
Figu e 5: E ec s o TCEP and EDTA on con o ma ion o MDMX-S403D FL.
(A) CD spec oscopy o MDMX-S403D FL measu ed in 10 mM sodium phospha e bu e (pH
7.5, wi hou NaCl) ac oss he wa eleng h ange 180–280 nm. (B) In insic luo escence
emission spec a o MDMX-S403D FL measu ed in RNA-binding bu e (150 mM NaCl,
20 mM T is-HCl, pH 7.5) upon exci a ion a 280 nm. In bo h (A) and (B), measu emen s we e
pe o med unde ou condi ions: un ea ed (black line), wi h 1 mM TCEP (black dashed line),
wi h 5 mM TCEP (g ey line), and wi h a combina ion o 1 mM EDTA and 1 mM TCEP ( ed
line).
Figu e S1: HDX-MS analysis o MDMX-S403D FL in e ac ion wi h TP53 mRNA.
HDX-MS analysis o ull-leng h MDMX ca ying a phosphomime ic mu a ion a se ine 403
(MDMX-S403D FL) in complex wi h TP53 mRNA. Rela i e ac ional up ake ac oss amino
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acid esidues is shown o ee MDMX-S403D FL (o ange) and he MDMX-S403D FL–TP53
mRNA complex (blue) a e 10 s (A) and 1800 s (B) o deu e ium exposu e. Non-
exchangeable p oline esidues a e indica ed as gaps in he p o iles.
Figu e S2: E ec o NaCl concen a ion on ee TP53 mRNA mobili y.
EMSA analysis o ee TP53 mRNA in binding bu e and ei he 150 mM o 600 mM NaCl, as
indica ed. No p o ein was added o he eac ion. RNA was isualized on aga ose gels by
GelRed s aining.
Figu e S3: Schema ic ep esen a ion o pH-dependen cha ge dis ibu ion in MDMX.
Schema ic ep esen a ion o p edic ed cha ge dis ibu ion changes along he MDMX p o ein
sequence a pH 7.0 e sus pH 8.0. Posi i ely cha ged esidues a e shown in blue, nega i ely
cha ged esidues in ed, and amino acids a ec ed by he pH shi a e highligh ed in yellow.
The amino acid sequence o MDMX and i s p edic ed s uc u al changes unde a ying pH
condi ions we e isualized using Pa chwo k (h ps://pa chwo k.biologie.uni- eibu g.de/). The
inal schema ic ep esen a ion was hen p epa ed in BioRende . A ailable om:
h ps://BioRende .com/9gxy j . Ag eemen numbe : ED28BFG4CD, License numbe :
66587182.
Figu e S4: Modula ion o TP53 mRNA–MDMX-S403D (322-490) in e ac ion by zinc,
EDTA, and educing condi ions.
(A) EMSA analysis o TP53 mRNA binding o MDMX-S403D (322-490) cons uc in he
p esence o Zn²⁺and EDTA. P o eins we e incuba ed wi h TP53 mRNA (lanes 2–5) o
wi hou RNA (lanes 6–8) in binding bu e (150 mM NaCl, 50 mM T is, 1 mM TCEP, pH 7.5)
and ei he 10 µM o 100 µM ZnSO₄, in he p esence o EDTA (do ed columns) o absence
o EDTA (emp y columns). F ee RNA (lane 1) se ed as a con ol. (B, D) Quan i ica ion o
ee RNA band in ensi y om gels shown in (A) and (C), espec i ely, no malized o hei
co esponding RNA-only con ol. Da a ep esen mean ± SEM (n = 3 biological eplica es; *p
< 0.05, **p < 0.01). Addi ional s a is ical compa isons a e shown in he igu e and
al e na i ely indica ed as •p < 0.05. (C) EMSA showing he e ec o he educing agen
TCEP on TP53 mRNA o MDMX-S403D (322-490) cons uc . P o eins we e incuba ed wi h
TP53 mRNA (lanes 2–4) o wi hou RNA (lanes 5–7) in binding bu e con aining 0, 5, o
10 mM TCEP. F ee RNA (lane 1) se ed as a con ol. In panels (A) and (C), b acke s
indica e smea s co esponding o p o ein–TP53 mRNA complexes. RNA–p o ein complexes
we e isualized by GelRed s aining (uppe panels), and o al p o ein was de ec ed using
SYPRO Ruby s aining o he same gels (lowe panels). The RNA:p o ein mola a io was
1:50 in all eac ions. (E) O e lay o EMSA gel s ained wi h GelRed and SYPRO Ruby
showing TP53 mRNA binding o MDMX-S403D FL in he p esence o Zn²⁺and EDTA,
co esponding o (A). The uppe band co esponds o he RNA–p o ein complex, while he
lowe band ep esen s unbound p o ein.
Figu e S5: E ec s o TCEP and EDTA on con o ma ion o MDMX-S403D (322–490).
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(A) CD spec oscopy o MDMX-S403D (322-490) measu ed in 10 mM sodium phospha e
bu e (pH 7.5, wi hou NaCl) ac oss he wa eleng h ange 180–280 nm. (B) In insic
luo escence emission spec a o MDMX-S403D FL measu ed in RNA-binding bu e
(150 mM NaCl, 20 mM T is-HCl, pH 7.5) upon exci a ion a 280 nm. In bo h (A) and (B),
measu emen s we e pe o med unde ou condi ions: un ea ed (black line), wi h 1 mM
TCEP (black dashed line), wi h 5 mM TCEP (g ey line), and wi h a combina ion o 1 mM
EDTA and 1 mM TCEP ( ed line). (C) CD spec oscopy o MDMX-S403D FL measu ed in
10 mM sodium phospha e bu e (pH 7.5, wi hou NaCl) ac oss a wa eleng h ange o 180–
280 nm. The spec a show he p o ein in he absence o EDTA (black line) and in he
p esence o 1 mM EDTA ( ed line). (D) EMSA analysis o TP53 mRNA binding MDMX-
S403D FL in he p esence o TCEP and EDTA. Reac ions we e ca ied ou a an
RNA:p o ein mola a io o 1:50 in binding bu e con aining 1 mM TCEP (lane 3), 1 mM
EDTA (lane 4), o a combina ion o bo h (lane 5). F ee RNA (lane 1) and un ea ed p o ein
(lane 2) se ed as con ols. RNA and RNA–p o ein complexes we e isualized on GelRed-
s ained aga ose gels (uppe panel), and o al p o ein was de ec ed using SYPRO Ruby
s aining o he same gel (lowe panel). (E) Quan i ica ion o ee RNA band in ensi y o
MDMX-S403 FL ea ed wi h only TCEP (emp y columns) and wi h a combina ion o TCEP
and EDTA (do ed columns), no malized o he RNA-only con ol. Da a ep esen mean ±
SEM (n = 3 biological eplica es; *p < 0.05, **p < 0.01). Addi ional s a is ical compa isons a e
shown in he igu e and al e na i ely indica ed as •p < 0.05 and •••p < 0.001.
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Figu e 1
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Figu e 2
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Figu e 3
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Figu e 4
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