W196 and the ß -Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor

Resea ch A icle W196 and he β-Hai pin Mo i Modula e he Redox Swi ch o Con o ma ion and he Biomolecula In e ac ion Ne wo k o he Apop osis-Inducing Fac o Sil ia Rome o-Tamayo, 1,2 Ruben Laplaza, 3,4 Ad ian Velazquez-Campoy , 1,2,5,6,7 Raquel Villanue a , 1,2 Milag os Medina , 1,2 and Pa icia Fe ei a 1,2 1 Depa amen o de Bioquímica y Biología Molecula y Celula , Facul ad de Ciencias, Uni e sidad de Za agoza, Spain 2 Ins i u o de Biocompu ación y Física de Sis emas Complejos, BIFI (GBsC-CSIC and BIFI-IQFR Join Uni s), Uni e sidad de Za agoza, Spain 3 So bonne Uni e si é, CNRS, Labo a oi e de Chimie Théo ique, LCT, 75005 Pa is, F ance 4 Depa amen o de Química Física, Uni e sidad de Za agoza, 50009 Za agoza, Spain 5 Fundación ARAID, Dipu ación Gene al de A agón, Spain 6 A agon Ins i u e o Heal h Resea ch (IIS A agon), Za agoza, Spain 7 Biomedical Resea ch Ne wo king Cen e o Li e and Diges i e Diseases (CIBERehd), Mad id, Spain Co espondence should be add essed o Milag os Medina; [email p o ec ed] and Pa icia Fe ei a; [email p o ec ed] Recei ed 4 No embe 2020; Re ised 9 Decembe 2020; Accep ed 18 Decembe 2020; Published 15 Janua y 2021 Academic Edi o : Luciana Hannibal Copy igh © 2021 Sil ia Rome o-Tamayo e al. This is an open access a icle dis ibu ed unde 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 wo k is p ope ly ci ed. The human apop osis-inducing ac o (hAIF) is a moonligh fla op o ein in ol ed in mi ochond ial espi a o y complex assembly and caspase-independen p og ammed cell dea h. These unc ions migh be modula ed by i s edox-linked s uc u al ansi ion ha enables hAIF o ac as a NAD(H/ + ) edox senso . Upon educ ion wi h NADH, hAIF unde goes a con o ma ional eo ganiza ion in wo specific inse ions— he flexible egula o y C-loop and he 190-202 β-ha pin—p omo ing p o ein dime iza ion and he s abiliza ion o a long-li e cha ge ans e complex (CTC) ha modula es i s monome -dime equilib ium and i s p o ein in e ac ion ne wo k in heal hy mi ochond ia. In his ega d, he e, we in es iga ed he p ecise unc ion o he β-hai pin in he AIF con o ma ion landscape ela ed o i s edox mechanism, by analyzing he ole played by W196, a key esidue in he in e ac ion o his mo i wi h he egula o y C-loop. Mu a ions a W196 dec ease he compac ness and s abili y o he oxidized hAIF, indica ing ha he β-hai pin and C-loop coupling con ibu e o p o ein s abili y. Kine ic s udies complemen ed wi h compu a ional simula ions e eal ha W196 and he β-hai pin con o ma ion modula e he low efficiency o hAIF as NADH oxido educ ase, con ibu ing o configu e i s ac i e si e in a noncompe en geome y o hyd ide ans e and o s abilize he CTC s a e by enhancing he affini y o NAD + . Finally, he β-hai pin mo i con ibu es o define he con o ma ion o AIF’s in e ac ion su aces wi h i s physiological pa ne s. These findings imp o e ou unde s anding on he molecula basis o hAIF’s cellula ac i i ies, a c ucial aspec o cla i ying i s associa ed pa hological mechanisms and de eloping new molecula he apies. 1. In oduc ion The human apop osis-inducing ac o (hAIF) was fi s desc ibed as a mi ochond ial- eleased fla op o ein media ing caspase-independen p og ammed cell dea h [1]. Mo eo e , his ubiqui ously exp essed p o ein ac oss euka yo es also plays a i al ole in cell de elopmen and su i al [2]. These su i al unc ions ely on i s FAD-dependen ac i i ies, which con ibu e o main ain he s abili y o he mi ochon- d ial elec on ans e chain, supe complex o ganiza ion, and ansmemb ane po en ial, as well as o con ol mi o- chond ial eac i e oxygen species (ROS) [3]. In heal hy mi o- chond ia, he hAIF is p ocessed and he hAIF Δ1-53 ma u e p o ein ancho s in he inne memb ane (IM)— ia i s N- Hindawi Oxida i e Medicine and Cellula Longe i y Volume 2021, A icle ID 6673661, 19 pages h ps://doi.o g/10.1155/2021/6673661 e minal segmen , acing he in e memb ane space (IMS)— and olds in h ee domains (Figu e 1(a)) [4–6]. Mammalian AIFs ha e wo specific inse ions, a egula o y C- e minal loop (aa 510-560 in hAIF) and a β-hai pin (aa 190-202 in hAIF), which connec he NADH and FAD domains o he C- e minal p oapop o ic domain (Figu e 1(a)). hAIF con o ma ion is dynamically influenced by coen- zyme subs a e binding and by he edox swi ch o i s fla in co ac o , ac s belie ed o modula e i s biomolecula in e ac- ion ne wo k [7, 8]. In oxidized hAIF (hAIF ox ), he egula o y C-loop is s abilized in he p o ein co e by di ec in e ac ion wi h he β-hai pin, pa icula ly h ough s acking and H- bonding in e ac ions o W196 and R201 esidues wi h i s 517-524 and 529-533 sho helixes. Binding o one NADH molecule o AIF’s ac i e si e (NADH A ) p omo es FAD educ ion, as well as he s abiliza ion o a long-li ed FADH - /NAD + cha ge ans e complex (CTC). This CTC is inefficien in elec on ans e , bu capable o inducing a edox-linked p o ein con o ma ional eo ganiza ion and i s subsequen dime iza ion. CTC o ma ion displaces he β- hai pin ha igge s C-loop emodeling and i s elease o he sol en . These con o ma ional changes induce (i) he allos e ic o ma ion o he second nonca aly ic NADH bind- ing si e (NADH B ), whe e s acking in e ac ions wi h eo - ien ed W196 and F582 side chains acili a e NADH B accommoda ion and (ii) he dime iza ion o he p o ein (Figu es 1(c) and 1(e)). These ac s led o pos ula e AIF as a edox senso o NAD(H/ + ) cellula le els [9–11]. W196 sub- s i u ion by alanine dis up s he in e ac ion be ween he β- hai pin and he C-loop ha unwinds he abo e men ioned 529-533 helix and eleases he wo specific AIF inse ions o he sol en , p omo ing a pe missi e mu an dime iza ion in i s oxidized s a e (W196A hAIF Δ1-101ox , he ein W196A ox ) (Figu es 1(a) and 1(d)) [11]. Howe e , W196A ox main ains an ac i e-si e a chi ec u e simila o ha o WT hAIF ox o esidues in ol ed in NADH A binding wi h he only excep- ions o E453 and H454 (Figu e 1(b)). The β-hai pin elease in W196A ox also induces he displacemen o he cen al β- s and and he eo ien a ion o E453 and H454 side chains (Figu e S1D-E). Thus, H454 dis up s i s in e ac ion wi h S480, p oducing as a consequence he displacemen o he H478 side chain—si ed in he loop connec ing he cen al β-s and and he His- ich helix— owa ds he C-loop, con ibu ing o i s elease, and he exposi ion o he hyd ophobic bo de a he dime iza ion in e aces in he W196A ox s uc u e. Such las con o ma ional changes a e simila o hose epo ed o he WT CTC s uc u e (Figu e S1E-F). In heal hy cells, hAIF is essen ial o mi ochond ial bio- ene ge ics, being i s physical and unc ional in e ac ion wi h human CHCHD4 (coiled-coil-helix-coiled-coil-helix domain con aining 4) key in he assembly and/o s abiliza- ion o mul isubuni espi a o y anspo chain complexes and supe complexes [12–15]. In IMS, CHCHD4 con ols he impo and oxida i e olding o a se o assembly ac o s and p o ein subuni s o espi a o y complexes, while hAIF would egula e CHCHD4 exp ession as well as i s impo and p ope IMS localiza ion. Consequen ly, down egula ion o deple ion o hAIF gi es ise o majo dys unc ions in oxi- da i e phospho yla ion (OXPHOS), seconda y o he defi- ciency o CHCHD4, causing se e e neu odegene a i e illnesses [12, 14, 16, 17]. The hAIF con o ma ion—modula ed by i s edox NADH-dependen monome -dime equilib iu- m—is sugges ed o be c i ical o his in e ac ion [7, 13, 18]. Upon le hal cellula s ess, hAIF ac s as a media o o nec o ic poly(ADP- ibose) polyme ase- (PARP-) 1- dependen cell dea h (pa hana os) by i s u he p ocessing in o he soluble p oapop o ic o m (hAIF Δ1-101 ) and i s elease in o he cy osol. The egula o y mechanism by which AIF is eleased is unknown, bu could be somehow modu- la ed by i s s uc u al eo ganiza ion due o deple ion o coenzyme le els du ing PARP-1 hype ac i a ion [19]. Once in he cy osol, i s in e ac ion wi h some endonucleases, as cyclophilin A (CypA), a o s nuclea co ansloca ion o he AIF:CypA complex [20, 21]. In his subcellula compa - men , he associa ion o his bina y complex o he his one H2AX leads o he assembly o he AIF-media ed DNA deg- ada ion complex (“deg adosome,”AIF:CypA:H2AX:DNA), which p o okes ch oma in condensa ion and DNA agmen- a ion [21, 22]. Despi e he eme ging pic u e o he physiological unc- ions o AIF being modula ed by i s con o ma ional and edox s a es, we a e only s a ing o depic he implica ions o he molecula mechanism egula ing i s ac i i ies. Thus, he molecula basis o he mechanism by which AIF egu- la es and pi o s he edox-dependen in e ac ion wi h CHCHD4, as well as hose o he ac ion o he deg adosome complex as a dea h effec o emain unknown. None heless, we can en isage ha AIF abili y o s abilize bo h s able CTC and dime s—upon in e ac ion wi h he coenzyme ollowed by FAD educ ion—is su ely a key ea u e o swi ch among i s in i o oles. In his con ex , he s uc u al changes induced by CTC o ma ion in na i e p o ein, bu also sha ed by W196A ox , sugges ha W196 and/o he β-hai pin migh be ele an o AIF cellula ac i i ies. Such hypo hesis is u - he suppo ed by he β-hai pin con ibu ing o binding o he allos e ic NADH B , as well as by he ac ha pa hogenic mu a ions cou sing wi h se e e p ocesses o neu odegene a- ion and ea ly dea h ha e been epo ed a bo h he NADH B binding si e and he β-hai pin i sel . In he p esen s udy, we pa icula ly in es iga e he con- ibu ion o he β-hai pin o he egula ion o hAIF s uc u al s abili y, coenzyme binding, educ ase ac i i y, CTC s abili y, and in e ac ion wi h i s physiological pa ne s, by gene a ing W196A, W196L, and W196Y si e-di ec ed mu an s (which p og essi ely educe a oma ic and s acking in e ac ions). Ou esul s indica e ha he W196 side chain is no only key o es ablish he β-hai pin and C-loop o ganiza ion in he oxidized s a e, bu also o egula e he s abili y and con- o ma ional landscape o he p o ein. Bo h ac s seem o be ele an o de e mine AIF efficiency as a cellula edox sen- so , as well as o he es ablishmen o specific bina y in e ac- ions wi h diffe en pa ne s. 2. Ma e ials and Me hods 2.1. Exp ession and P oduc ion o P o eins. The cDNA sequences encoding o W196Y, W196L, and W196A 2 Oxida i e Medicine and Cellula Longe i y Helix 517-524 Helix 529-533 𝛽-hai pin FAD C-loop (a) W483 F310 E314 L311 G308 G339 E336 H454 E453 (b) W483 H454 F310 E314 L311 G308 G339 E336 E453 NADA + (c) W483 W196A E493 F582 (d) Figu e 1: Con inued. 3Oxida i e Medicine and Cellula Longe i y hAIF Δ1-101 a ian s (UniP o KB O95831) we e ob ained by si e-di ec ed mu agenesis om Mu agenex® and hen sub- cloned in o he pET28a exp ession ec o wi h a clea able N- e minal His 6 - ag simila o ha epo ed o he WT p o- ein [10]. The cDNAs encoding o human CypA (Uni- P o KB P62937), CHCHD4 (UniP o KB Q8N4Q1), and His one H2AX (UniP o KB P16104) we e syn he ized wi h a clea able N- e minal His 6 - ag (CACCAT) and codon op i- mized o Esche ichia coli exp ession by GenSc ip ®. The coding sequences we e subcloned in o he pET28a exp ession ec o be ween wo es ic ion si es: NdeI-No I o CypA and CHCHD4 and NcoI-NdeI o H2AX. The esul ing con- s uc s we e used o ans o m he E. coli C41 (DE3) s ain o he e ologous p o ein exp ession. P o eins we e exp essed and pu ified as desc ibed in he supplemen a y ma e ials. 2.2. Molecula Weigh De e mina ion by Size Exclusion Ch oma og aphy. The hAIF Δ1-101 a ian s, ei he in he p es- ence o absence o a 10- old excess o NADH, we e loaded on o a HiP ep 26/60 Sephac yl™S-200 High Resolu ion (GE Heal hca e, Chicago, IL) column a ached o a as p essu e liquid ch oma og aphic sys em (GE Heal hca e, Chicago, IL). P o ein elu ion was pe o med in 50 mM phospha e buffe , 150 mM NaCl, pH 7.4, a a flow a e o 0.5 mL/min. The column was p e iously calib a ed wi h he GE Heal h- ca e LMW calib a ion ki (6 p o eins in he 6400- 160000 Da ange). The ob ained ch oma og ams we e fi ed o a se o Gaussian unc ions. 2.3. S abiliza ion o C oss-Linked P o ein Oligome s and Elec opho e ic Analysis. Reac ion mix u es con aining 4 μM o he hAIF Δ1-101 a ian s in 10 mM phospha e, pH 7.4 we e incuba ed wi h a 100- old excess o he homobi unc ional- bis[sul osuccinimidyl]-sube a e (BS 3 ) (Pie ce) c oss-linke a oom empe a u e in he absence o p esence o a 10- old excess o NADH. Reac ions we e s opped by he addi ion o he dena u ing b omophenol blue sample buffe and hea ed 5 min a 95 ° C. Sample mix u es we e hen esol ed by 12% SDS-PAGE. 2.4. Spec oscopic Cha ac e iza ion. UV- isible spec a we e eco ded in a Ca y 100 Bio spec opho ome e (Agilen , San a Cla a, CA). P o ein concen a ions we e de e mined using he mola abso p ion coefficien s o each a ian , which we e es ima ed by p o ein dena u a ion wi h 3 M guanidi- nium chlo ide in 10 mM phospha e, pH 7.4, ollowed by quan ifica ion o he eleased FAD. The ex inc ion coeffi- cien s o WT, W196A, W196L, and W196Y hAIF Δ1-101ox we e ε451nm =13:7M −1cm−1[10], ε451nm =13:35 M−1cm−1, ε451nm =13:92 M−1cm−1, and ε452nm =14:01 M−1cm−1 espec i ely. Ci cula dich oism (CD) spec a we e eco ded in a he mos a ed Chi ascan (Applied Pho ophysics L d., Su - ey, UK). Fa -UV CD spec a we e acqui ed using 1 μM p o- ein in a 0.1 cm pa hleng h cu e e, while nea -UV/Vis CD spec a we e eco ded using 20 μM p o ein in a 1 cm pa h- leng h cu e e. Fluo escence spec a we e eco ded in a he mos a ed Ca y Eclipse Fluo escence spec opho ome e (Agilen , San a Cla a, CA) using 2 μM p o ein in a 1 cm pa h- leng h cu e e. Fla in fluo escence emission spec a we e acqui ed in he 480-600 nm ange upon exci a ion a 450 nm. Fluo escence emission spec a o a oma ic esidues we e collec ed om 300 o 550 nm upon exci a ion a 280 nm. CD and fluo escence spec a we e eco ded in he absence and p esence o a 100- old excess o NADH a 10 ° C ( olded s a e) and 90 ° C ( he mally dena u ed s a e). W483 W196A E493 F582 NADHB (e) Figu e 1: Compa a i e o e iew o he c ys allog aphic s uc u es o WT hAIF Δ1-101ox (PDB 4BV6), WT CTC hAIF Δ1-101 d :2NAD( + /H) (PDB 4BUR), and W196A ox a ian (PDB 5KVH). (a) Ca oon supe posi ion. FAD-, NADH-, and C- e minal domains colo ed in gold, ligh blue, and pale g een, espec i ely. FAD d awn as s icks wi h C a oms in yellow, salmon, and magen a, espec i ely, o WT ox ,WT CTC, and W196A ox s uc u es. Visible esidues in he β-hai pin and he egula o y 509-560 C-loop a e shown in ed, magen a, and salmon, espec i ely, o WT ox , WT CTC, and W196A ox s uc u es. Missing agmen s o he C-loop (P545-D559, K518-G557 and A511- D559 in WT ox chain A, WT CTC chain C, and W196A ox chain A s uc u es, espec i ely) a e indica ed as dashed lines. De ail o he W196A ox NADH A binding si e o e laid wi h (b) WT ox and (c) WT CTC. De ail o he W196A ox NADH B binding si e o e laid wi h (d) WT ox and (e) WT CTC. Side chains o ele an esidues a e shown as CPK colo ed s icks wi h C a oms in salmon o W196A ox and in ligh g ey o WT ox and WT CTC s uc u es. NAD( + /H) A and NADH B in he WT CTC s uc u e a e shown as CPK colo ed s icks wi h i s C a oms in blue. 4 Oxida i e Medicine and Cellula Longe i y 2.5. The mal Dena u a ion Assays. The mal dena u a ion cu es we e ollowed by changes in he FAD fluo escence emission upon i s elease om he p o ein by sample exci a- ion a 450 nm. Cu es we e moni o ed om 10 ° C o90 ° C wi h scan a es o 1 ° C/min, bo h in he absence and p esence o a 100- old excess o NADH. The cu es o each a ian we e oughly no malized o alues be ween 0 and 1 and glob- ally fi ed o a wo-s ep p ocess desc ibing a single ansi ion un olding equilib ium (na i e (N)↔un olded (U)) by using he ollowing equa ion [23]: Sobs =SN+mNT+SU+mUT ðÞ e−ΔG/RT ðÞ 1+e−ΔG/RT ðÞ ,ð1Þ in which Sobs is he measu ed p o ein signal a a gi en em- pe a u e (T). SNand SUa e in e cep a 0 K wi h he y-axis o he linea ex apola ion o he na i e and un olded p e- and pos ansi ion egions, espec i ely, while mNand mU a e he co esponding slopes. The s abiliza ion Gibbs ene gy depends on empe a u e acco ding o ΔG=ΔHð1−1/TmÞ +ΔCPðT−Tm−Tln ðT/TmÞÞ, whe e ΔHis he un olding en halpy, Tmis he mid ansi ion empe a u e, ΔCPis he un olding hea capaci y change, and Ris he ideal gas cons an . 2.6. Kine ics Measu emen s. The s eady-s a e diapho ase ac i i y o hAIF Δ1-101 a ian s was measu ed in ai sa u a ed 50 mM po assium phospha e, pH 8.0, using NADH as he subs a e dono and 95 μM dichlo ophenolindophenol (DCPIP, Δε620nm =21mM −1cm−1) as accep o [10]. When sa u a ion p ofiles on he py idine nucleo ide concen a ion we e obse ed, kine ic cons an s we e es ima ed by fi ing ini ial eac ion a es a diffe en coenzyme concen a ions o he Michaelis-Men en equa ion: ν e=kca NADH ½ KNADH m+ NADH ½ , ν e=kca /KNADH mNADH ½ 1+ kca /KNADH mNADH ½ /kca  , ð2Þ whe e s ands o he ini ial eloci y, eis he enzyme concen- a ion, KNADH mis he Michaelis cons an o he enzyme- NADH complex, kca is he u no e numbe o he enzyme, and kca /KNADH mis he enzyme ca aly ic efficiency. The eac i i y o he CTC owa ds molecula oxygen was moni o ed by ull educ ion o hAIF Δ1-101 samples wi h NADH (1.5- old he concen a ion o he p o ein) in 50 mM phospha e buffe , pH 7.4, and ollowing hei eoxida- ion in a Ca y 100 spec opho ome e (Agilen , San a Cla a, CA). Abso p ion spec a we e eco ded a 25 ° C un il ull oxi- da ion o he fla in co ac o was achie ed. Fo each ime, he pe cen emaining o CTC e sus eoxida ion by molecula oxygen was es ima ed as ΔA /ΔAmax, whe e ΔAmax is he di - e ence be ween he minimum and he maximum abso - bance a 700 nm, and ΔA is he diffe ence o each alue a 700nm minus he minimum abso bance a 700 nm. The CTC hal -li e is he ime a which 50% o CTC s ill emains. A SX18.MV s opped-flow spec opho ome e (Applied Pho ophysics L d., Su ey, UK), in e aced wi h he P oDa a-SX so wa e and a pho odiode a ay de ec o , was used o in es iga e he as kine ic educ ion o he hAIF a - ian s by he NADH coenzyme. Samples o ~10 μM hAIF Δ1- 101ox we e mixed wi h inc easing concen a ions o NADH (0.03-10 mM) unde ae obic condi ions in 50 mM po assium phospha e, pH 7.4, a 25 ° C. The enzyme and NADH concen- a ions a e he final ones ob ained a e mixing equal ol- umes o subs a e and enzyme. Obse ed a e cons an s o he hyd ide ans e (HT) e en (kobs) we e calcula ed by global analysis and nume ical in eg a ion me hods (simul a- neously using all spec al da a in he 400-800 nm egion along ime e olu ion). A single-s ep model (A→B) bes fi ed o desc ibe he o e all eac ion a all NADH concen a ions assayed. A e aged kobs alues a each NADH concen a ion we e hen fi ed o he equa ion ha desc ibes he o ma ion o an enzyme:subs a e complex p io o he HT e en : kobs =kHTNADH KNADH d+ NADH +k e ,ð3Þ whe e kHT is he limi ing a e cons an o HT om he py - idine nucleo ide coenzyme o he FAD co ac o o hAIF, KNADH dis he dissocia ion cons an o he ansien hAIF Δ1- 101ox :NADH complex, and k e is he eac ion cons an o a po en ial o e all e e se p ocess. S opped-flow spec opho ome y was also used o e alu- a e he a e cons an s o CTC o ma ion when mixing pho o- educed hAIF Δ1-101 (hAIF Δ1-101ph d ) wi h inc easing concen a ions o NAD + (0.125-5 mM) unde anae obic con- di ions. hAIF Δ1-101ph d samples we e ob ained by pho o e- duc ion in he p esence o 5 μM me hyl iologen, 3 μM5- deaza ibofla in, and 20 mM EDTA. The assays we e pe - o med a 25 ° C in 50 mM po assium phospha e, pH 7.4, unde anae obic condi ions (ob ained by se e al cycles o acuum applica ion and bubbling wi h O 2 ee a gon). Da a we e global fi ed o a single s ep model (A→B), and kobs we e de e mined a he diffe en NAD + concen a ions assayed. These alues we e hen fi ed o he equa ion ha desc ibes he o ma ion o a ansien hAIF Δ1-101ph d :NAD + complex p io o he CTC s abiliza ion: kobs =kCTC NAD+ ½ KNAD+ d+ NAD+ ½ ,ð4Þ in which kCTC is he limi ing a e cons an o he ea ange- men o he encoun e complex o o m he CTC, and KNAD+ d is he dissocia ion cons an o he men ioned ansien encoun e complex. 2.7. Iso he mal Ti a ion Calo ime y (ITC). ITC assays we e ca ied ou using an Au o-iTC200 (Mic oCal,Mal e n-Pana- ly ical, Mal e n, UK) he mos a ed a 25 ° C. Typically, 10- 20 μM p o ein pa ne and dsDNA samples—p epa ed as desc ibed below—we e used o i a e ~10 μM hAIF Δ1-101 a ian s. All solu ions we e degassed a 15 ° C o 1 min be o e each assay. A sequence o 2 μL injec ions o i an solu ion 5Oxida i e Medicine and Cellula Longe i y e e y 150 s was p og ammed, and he s i ing speed was se o 750 pm. The associa ion cons an (Ka), he en halpy o binding (ΔH), and he binding s oichiome y (N) we e es i- ma ed h ough nonlinea leas -squa es eg ession o he expe imen al da a employing a single-ligand binding si e model implemen ed in O igin 7.0 (O iginLab, No hamp on, MA). The dissocia ion cons an (Kd), he ee ene gy change (ΔG), and he en opy change (ΔS) we e ob ained om basic he modynamic ela ionships. Since hAIF binds DNA unspecifically, a 0.5 mM dsDNA sample was p epa ed om 1 mM solu ions o HPLC- pu ified o wa d and a e e se complemen a y 15-bp oligo- nucleo ides (5′- GGT TAG TTA TGC GCG -3′; andomly designed) syn he ized by In eg a ed DNA Technologies. The pai o oligonucleo ides was mixed a an equimola a io and annealed by hea ing 1 min a 99 ° C and pe o ming a 3 h empe a u e scanning om 95 o 25 ° C, dec easing 1 ° C each 3 min. 0.5 mM dsDNA s ock solu ions we e ob ained. 2.8. Gene a ion o S uc u al Models. Models con aining he missing C-loop esidues (546–558 and 518-559, espec i ely, o c ys al s uc u es o WT hAIF Δ1-101ox and hAIF Δ1- 101 d :NAD + s a es), as well as W196A, W196L, and W196Y mu a ions, we e buil using as empla es, he coo dina es o WT hAIF Δ1-101ox (PDB 4BV6) and hAIF Δ1-101 d :NAD + (PDB 4BUR) and he Swiss-Model se e [7, 10, 24]. Rou- ines o minimiza ion and molecula dynamics (MD) simu- la ions ollowed p e ious epo ed p o ocols [7] and a e summa ized in he supplemen a y ma e ials. Imp o emen s include using a ime s ep o 2 s and pe o ming fi e eplicas o 10 ns MD p oduc ion o each model s uc u e. 2.9. Da a Analysis. Da a we e fi and shown using SigmaPlo (Sys a . So wa e Inc. Richmond, CA, USA), O igin 7.0 (O i- ginLab Co po a ion, No hamp on, MA), and P o-K (Applied Pho ophysics L d., Su ey, UK). VMD [25] and PyMol [26] we e used o analyze and isualize s uc u al da a, as well as o p oduce s uc u al figu es. 3. Resul s and Discussion 3.1. Mu a ions a W196 Residue Ha dly Impac s he O e all hAIF Δ1-101 Co e Con o ma ional P ope ies in Oxidized and NADH-Reduced S a es. The h ee W196 a ian s he e s udied we e pu ified o homogenei y as holop o eins a e hei exp ession in E.coli as desc ibed p e iously o he WT p o- ein [10]. Thei UV- isible abso p ion spec a showed he cha ac e is ic bands I and II o he fla in a 451 and 380 nm, espec i ely, a shoulde a 476 nm, and A280/A451 a io ≈11, indica ing ha , simila ly o he WT p o ein, he co ac o was in he oxidized s a e and co ec ly inco po a ed o he p o ein (Figu e S2A). Only W196A showed a dis o ed shape o band II and lowe A451/A380 a io eflec ing some diffe ences in he en i onmen o i s fla in ing. The W196 a ian s also had simila a -UV CD spec a o he WT p o ein, wi h minima a ~222 and ~208 nm indica- i e o high α-helix con en (Figu e S2B). Reduc ion o he FAD co ac o by NADH p oduced he dec ease in ela i e in ensi y o minima a 208 nm o all mu an s (Figu e S2C), as p e iously epo ed o he WT p o ein [7]. This sugges s simila o e all con o ma ions in he CTCs. The nea - UV/Vis CD spec a o he a ian s showed he WT cha ac e is ic maxima (~300 nm and ~365 nm) and minima (~453 and ~477 nm) (Figu e S2D). Finally, changes obse ed upon incuba ion wi h NADH we e also consis en wi h FAD educ ion (lack o nea -UV CD signal a 300 nm and in he 350-500 nm ange) and CTC s abiliza ion (new minima a ~405 nm and b oad bands a ~600 nm) in all a ian s (Figu e S2E) [7]. Since he c ys al s uc u e is only a ailable o W196A ox , we buil s uc u al models con aining he W196 mu a ions, as well as he missed C-loop esidues in he WT X- ay s uc- u es, o u he e alua e he impac o mu a ions on he con- o ma ion o hAIF Δ1-101ox and i s CTC [7, 10]. Models o oxidized a ian s, including W196A ox , we e buil using he WT ox c ys al s uc u e as a empla e o be e e alua e he effec o each mu a ion on na i e s uc u es, hus p e en ing he o he a ian ’s models om being “ o ced” o beha e as W196A ox . A e 10 ns MD elaxa ion, only small fluc ua ions wi hin each simula ed sys em we e de ec ed o a e aged alues o ene gy, adius o gy a ion, RMSD, and sol en accessible su ace (SAS) o ligands, as well as o he main in e ac ions coupling he FAD co ac o and NADH coen- zyme o he p o ein (Figu es S3A and S4). These obse a ions con as wi h hose ob ained when simila ly e alua ing he pa hogenic dele ion o esidue R201 si ua ed oge he wi h W196 in he β-hai pin and also con ibu ing o C-loop linking [7]. This clinical ΔR201 a ian apidly b eaks he ne wo k linking he FAD co ac o , he β-hai pin i sel , he ac i e si e esidues, he cen al β-s and, and he C-loop du ing he MD p oduc ion [7]. Al oge he , expe imen al and modelling e idences indica e ha subs i u ions a W196 e ain he WT hAIF Δ1-101 a chi ec u e a he ac i e si e and he p o ein co e, in bo h he oxidized and CTC s a es. In ag eemen , W196A ox was e en able o c ys allize [11]. 3.2. W196 Side Chain Modula es he Monome -Dime Equilib ium in hAIF Δ1−101 .Gel fil a ion ch oma og aphy was used o s udy he impac o mu a ions on he abili y o hAIF Δ1−101 o unde go NADH-linked dime iza ion. While, simila ly o he WT ox p o ein (Figu e 2(a)) [10], he W196Y ox mu an elu ed as a monome o appa en molecu- la weigh ( app MW) ~45-58 kDa (Figu e 2(b)), he W196L ox and W196A ox a ian s elu ed as conside ably b oad peaks wi h lowe exclusion olumes. Peak decon olu ion sugges ed wo popula ions wi h app MW o 63 and 115 kDa o W196L ox and 75 and 138 kDa o W196A ox (Figu es 2(c) and 2(d), espec i ely), indica ing less compac monome ic con o ma- ions and/o a quick monome -dime exchange. Upon incu- ba ion wi h NADH, he W196Y and W196L a ian s elu ed mainly as a new peak o lowe exclusion olume (~145- 155 kDa) (Figu es 2(b) and 2(c)) ha was p e iously ela ed o he CTC dime in he WT p o ein (Figu e 2(a)). Finally, he elu ion peak o W196A in he p esence o NADH, when compa ed o W196A ox , also ge s na owe and sligh ly dis- placed owa ds he WT CTC dime elu ion olume (Figu e 2(d)). 6 Oxida i e Medicine and Cellula Longe i y Chemical c oss-linking wi h BS 3 —able o co alen ly con- juga e hAIF dime s bu no monome s— ollowed by assess- men o species by SDS-PAGE (Figu e 2(e)), was hen used o e alua e whe he he obse ed ch oma og aphic changes migh ela e o W196 mu a ions influencing he compac ness o p o ein con o ma ion and/o he CTC dime li e ime. Upon incuba ion wi h BS 3 , all oxidized mu an s exhibi ed he band o ∼55 kDa co esponding o he hAIF Δ1−101ox monome , al hough i was in gene al mo e diffuse han in he c oss-linke absence. When a ian s we e p eincuba ed wi h bo h NADH and BS 3 , an addi ional b oad band o ~170 kDa was de ec ed. In WT hAIF Δ1−101 , his band is ela ed o he p o ein abili y o unde go dime iza ion in he CTC s a e upon NADH binding and fla in educ ion [10]. No iceably, his band, indica i e o dime s abiliza ion, was also obse ed o W196A ox (in he absence o he coenzyme), in ag eemen wi h he exclusion ch oma og aphy da a ob ained o his a ian (Figu e 2(d)) and wi h i s epo ed dime ic c ys al s uc u e [11]. These da a confi m ha all W196 a ian s a e able o dime ize upon NADH educ ion, bu also show ha he mu a ions modula e he CTC dime s abili y. They also sugges con o ma ional changes ha a o he displacemen o he monome -dime equilib ium owa ds he dime in he oxidized s a e, pa icula ly in W196A ox . 3.3. W196 Highly Con ibu es o Modula e he Low Efficiency o hAIF Δ1-101 as NADH Oxidase. Unde physiological Elu ion olume (mL) 0 3 6 9 12 15 18 Abs280nm 0 20 40 60 80 Elu ion olume (mL) 0 3 6 9 12 15 18 0 20 40 60 80 100 Elu ion olume (mL) 0 3 6 9 12 15 18 Abs280nm 0 20 40 60 80 Elu ion olume (mL) 0 3 6 9 12 15 18 0 20 40 60 80 55 72 95 150 kDa WT W196Y W196L W196A −+ + −− + BS3 NADH −+ + −− + −+ + −− + −+ + −− + (a) (b) (c) (d) (e) Figu e 2: Effec o W196 eplacemen on he hAIF Δ1-101 abili y o s abilize dime s. Elu ion p ofile o (a) WT, (b) W196Y, (c) W196L, and (d) W196A on a Sephadex S-200 column a 6 ° C. The assays we e pe o med in absence and p esence o a 10- old excess o NADH, and p ofiles a e, espec i ely, shown in black con inuous and dashed lines. The espec i e diffe en popula ions assigned by Gaussian analysis a e depic ed in g ey lines. (e) Chemical c oss-linking o hAIF Δ1-101 samples (~3μM p o eins) wi h a 100- old excess o he BS 3 c oss-linke in he absence and p esence o NADH (300 μM). A e 45 minu es o incuba ion, he eac ions we e s opped by he addi ion o b omophenol sample buffe and esol ed by 12% SDS-PAGE. 7Oxida i e Medicine and Cellula Longe i y condi ions, hAIF exhibi s a NADH oxidase ac i i y ha can be in i o moni o ed using he s eady-s a e DCPIP- dependen diapho ase eac ion. When e alua ed in his way, all W196 a ian s showed highe u no e a es han he WT p o ein (~3- old inc ease o W196Y and W196L and ~5- old o W196A) (Table 1). Rega ding Km NADH, he W196Y a ian alue was simila o ha o he WT, while he W196L and W196A a ian s showed a significan dec ease (~3- and 10- old, espec i ely). Thus, W196Y, W196L, and W196A a ian s we e ~3, ~8, and~45 imes mo e efficien oxidizing NADH han he WT p o ein. None- heless, despi e hese W196 a ian s a e mo e efficien as oxi- do educ ases han WT hAIF Δ1−101 , hey we e unable o oxidize NADH when using molecula oxygen as elec on accep o , analogously o he WT p o ein [10]. In he ligh o hese esul s, we s udied he impac o he W196 mu a ions on he HT eac ion om NADH o he FAD co ac o o hAIF Δ1-101 by using s opped-flow ansien kine ics. The kine ic aces eco ded o all a ian s a diffe en NADH concen a ions indica ed an essen ially i e e sible wo- elec on educ ion o he FAD co ac o and he concomi an o ma ion o a long wa eleng h b oad band ela ed o he s a- biliza ion o he hAIF Δ1-101 d :NAD + CTC species (Figu e 3 and S5). The in ensi y o his CTC band (a ea in he 510 −800 nm egion minus ha o he ee p o ein) o W196A and W196Y a ian s was in he ange o ha obse ed o he WT [10], sugges ing simila pe cen age o CTC s abiliza- ion. Howe e , he lowe in ensi y o W196L CTC band (∼76%) indica es ei he diffe en cha ge dis ibu ion be ween coenzyme and FAD ings in he CTC (sugges i e o diffe en CTC geome y) o educ ion o he amoun o he CTC s a- bilized. In all cases, global analyses o he spec al ange ime e olu ions bes fi ed o a one-s ep model (A→B). Thus, he obse ed p ocesses appea ed including he as o ma ion o he ansien hAIF Δ1-101 :NADH eac i e complex ollowed by he HT eac ion and he CTC o ma ion (Scheme 1). As a consequence, he con o ma ional swi ches in β-hai pin and C-loop induce p o ein dime iza ion in W196 a ian s, wi h he po en ial excep ion o W196A ha p esumably migh be mos ly a dime wi h he C-loop al eady eleased in he oxidized s a e [11]. The kobs alues ob ained showed hype bolic dependence on NADH concen a ion o all a - ian s, allowing kHT and Kd NADH de e mina ion upon fi ing o he equa ion (4) (Figu e 3(e) and Table 1). All a ian s showed as e HT a e cons an s and highe affini y o he NADH subs a e han he WT p o ein (up o ~29- and 5- old, espec i ely, o W196A). Consequen ly, W196Y, W196L, and W196A we e ~12-, ~24-, and up o ~153- old mo e efficien han he WT enzyme as hyd ide accep o s om NADH. No iceably, and, con a y o ha desc ibed o he WT p o ein, all hese W196 a ian s showed kHT alues highe han hei u no e a es, sugges ing ha o hem, he HT eac ion is no he limi ing s ep du ing ca aly- sis. The e o e, he W196 side chain highly con ibu es o modula e he p ope ies o hAIF Δ1-101 as a nonefficien NADH oxidase. S uc u ally, W196 does no o m pa o he p o ein edox ac i e si e i sel . Howe e , W196 side chain s acks o P488 a he edge o he cen al β-s and, con ibu ing o si u- a e he β-hai pin and he C-loop o ming a ca i y a he bo - om o which si s W483—a esidue ha flanks he py imidine ing o FAD—(Figu e 1(b)). W196A mu a ion inc eases W483 sol en accessibili y and C-loop and β-hai pin flexibil- i y, a o ing hei displacemen om he WT ox posi ions (Figu es 1(c)–1(e) and 4). No iceably, we obse ed he β- hai pin displacemen om P488 as well as he cen al β- s and e ac ion om he beginning o ou W196A ox model MD ajec o ies (s a ing om WT ox s uc u e) (Figu e 5(a)). Howe e , he Y196 and L196 side chains con ibu e o main- ain β-hai pin posi ion in he W196Y ox and W196L ox ajec- o ies, and e ac ion o he cen al β-s and is ha dly deduced o W196L ox (Figu e 5(a)). T ajec o ies also show a la ge inc ease in he SAS o he β-hai pin o W196A ox el- a i e o he o he wo a ian s and he WT (Figu e S3B). Thus, MD simula ions p edic an inc ease in dis ances be ween W483 and a omic posi ions a he ac i e si e o he oxidized a ian s (Figu e S4A). Such changes in W483 sol en accessibili y and ac i e si e comp ession mus impac subs a e affini y and coupling in o a compe en complex o HT, as well as he FAD midpoin educ ion po en ial and/o elec onic dis ibu ion—as was p e iously epo ed o he mu ine W196A a ian (70 mV highe edox po en ial han hose o he WT p o ein)—[9]. In ag eemen , kine ic pa ame e s show W196A as he a ian diffe ing mo e om he WT beha io ega ding efficiency o bo h HT and NADH binding, ollowed—by a —by W196L and being he a oma ic subs i u ion he one p oducing a milde effec . Dynamics o ac i e si e in CTCs show highe flexibili y ega ding oxidized s a e (Figu e S4A-B), bu WT CTC keeps i s cha ac e is ic Table 1: S eady-s a e and p e-s eady-s a e kine ic pa ame e s o WT hAIF Δ1-101 and i s W196 a ian s. hAIF S eady-s a e P e-s eady-sa e kca (s -1 ) KNADH m (μM) kca /KNADH m (s -1 mM -1 ) kHT (s -1 ) KNADH d (μM) kHT/KNADH d (s -1 mM -1 ) kCTC 1 (s -1 ) KNAD+ d (μM) kCTC/KNAD+ d (s -1 mM -1 ) WT 0:9±0:1 495 ± 170 1:9±0:91:5±0:1 4090 ± 300 0:4±0:1 45 ± 2 2080 ± 250 22 ± 3 W196Y 2:7±0:1 505 ± 35 5:3±0:6 12 ± 1 2870 ± 320 4:0±0:8 16 ± 1 183 ± 27 87 ± 14 W196L 2:8±0:1 187 ± 18 15 ± 1:9 36 ± 1 1725 ± 210 21 ± 3 29 ± 2 433 ± 11 67 ± 18 W196A 4:3±0:2 25 ± 4 172 ± 35 126 ± 1 1070 ± 40 117 ± 5:3 30 ± 1 394 ± 50 76 ± 10 Assays we e pe o med a 25 ° C in 50 mM po assium phospha e, pH 7.4 (n=3, mean ±SD). 1 Kine ic pa ame e s o CTC o ma ions we e ob ained wi h hAIF Δ1-101ph d a ian s. 8 Oxida i e Medicine and Cellula Longe i y 400 500 600 700 800 𝜀 (mM−1 cm−1) 0 4 8 12 16 Time (s) 0246 Concen a ion (𝜇M) 0 2 4 6 400 500 600 700 800 0 4 8 12 16 Time (s) 0.00 0.02 0.04 0 2 4 6 𝜀 (mM−1 cm−1) Concen a ion (𝜇M) Abs750nm Time (s) 0.0 0.1 0.2 0.3 0.000 0.006 0.012 0.018 0.024 Time (s) 0.0 0.1 0.2 0.3 Abs750nm 0.000 0.012 0.018 0.024 0.030 Wa eleng h (nm) 400 500 600 700 800 Abso bance 0.00 0.03 0.06 0.09 AB 0.8 s−1 AB Wa eleng h (nm) 400 500 600 700 800 0.00 0.03 0.06 0.09 114 s−1 AB 25 s−1 Wa eleng h (nm) 400 500 600 700 800 Abso bance 0.00 0.04 0.08 0.12 AB 27 s−1 Wa eleng h (nm) 400 500 600 700 800 0.00 0.04 0.08 0.12 [NADH] (mM) 036912 0.0 0.4 0.8 1.2 kobs (s−1) kobs (s−1) WT 0 25 50 75 100 125 [NAD+] (mM) 0123456 0 7 14 21 28 35 (a) (b) (c) (d) (e) ( ) Figu e 3: Kine ic cha ac e iza ion o W196 hAIF Δ1-101 a ian s. Spec al e olu ion o he educ ion o (a) WT (~10 μM) and (b) W196A a ian (~10 μM) when mixed wi h NADH (2 and 5 mM o WT and W196A, espec i ely). Spec a o he educ ion o WT ox a e shown a 0.15, 1.05, 2.1, 4.2, 6, 10.05, 12.45, 20.1, 30, 40.05, and 50.1 s a e mixing and hose o W196A a 0.005, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.45, 0.5, and 0.55 s. Do ed lines co espond o he spec a o oxidized enzymes be o e mixing wi h he coenzyme. The co esponding inse s show he abso bance spec a o he in e media e species ob ained by fi ing he spec al e olu ion o a single s ep model (A→B) and he e olu ion o he concen a ion o each species. Kine ics o CTC o ma ion upon mixing o he hAIF Δ1-101ph d o ms o (c) WT and (d) i s W196A a ian wi h NAD + (5 mM) unde anae obic condi ions. Spec al e olu ion o CTC o ma ion o WT a 0.001, 0.005, 0.01, 0.015, 0.02, 0.07, 0.1, 0.15, 0.2, 0.33, 0.4, and 0.5 s a e mixing and hose o W196A a 0.001, 0.002, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.03, 0.05, 0.07, 0.09, 0.1, 0.3, and 0.5 s. The co esponding inse s show he abso p ion e olu ion a 750 nm (black ci cle) and he fi s (con inuous line) a his wa eleng h a e globally fi ing e olu ion a a single s ep model (A→B). (e) Dependence o he obse ed a e cons an s o fla in educ ion o he WT (black ci cle), W196Y (black iangle), W196L (black diamond), and W196A (g ey squa e) eac ions on he NADH concen a ion. Lines ep esen he fi s o expe imen al da a o equa ion (3). ( ) Dependence o obse ed a e cons an s o CTC o ma ion when using WT (black ci cle), W196Y (black iangle), W196L (black diamond), and W196A (g ey squa e) hAIF Δ1-101ph d on he NAD + concen a ion. Lines ep esen he fi o expe imen al da a o equa ion (4). Assays we e pe o med in a s opped-flow spec opho ome e in 50 mM po assium phospha e, pH 7.4, and a 25 ° C(n=3,mean ± SD). 9Oxida i e Medicine and Cellula Longe i y [CHCHD4]T/[WT hAIF𝛥1-101 d:NAD+]T 0.0 0.5 1.0 1.5 2.0 –20.0 –15.0 –10.0 –5.0 0.0 5.0 –0.15 –0.10 –0.05 0.00 0.05 0 1020304050 Time (min) dQ/d (𝜇cal/s)Q (kcal/mol o injec an ) (a) [CHCHD4]T/[W196A hAIF𝛥1-101 d:NAD+]T 0.0 0.5 1.0 1.5 2.0 –8.0 –6.0 –4.0 –2.0 –0.06 –0.04 –0.02 0.00 0.02 0 1020304050 Time (min) dQ/d (𝜇cal/s) Q (kcal/mol o injec an ) (b) [CHCHD4]T/[W196A hAIF𝛥1-101ox]T 0.0 0.5 1.0 1.5 2.0 16 20 24 28 –0.2 0.0 0.2 0.4 0.6 0.8 0 1020304050 Time (min) dQ/d (𝜇cal/s)Q (kcal/mol o injec an ) (c) 0.0 0.5 1.0 1.5 2.0 –10.0 –8.0 –6.0 –4.0 –0.06 –0.04 –0.02 0.00 0.02 01020304050 Time (min) dQ/d (𝜇cal/s) [CHCHD4]T/[W196A hAIF𝛥1-101 d:NAD+]T Q (kcal/mol o injec an ) (d) Figu e 7: Effec o he W196 eplacemen in he binding o CHCHD4 o hAIF Δ1-101 . Calo ime ic i a ions o (a) WT CTC, (b) W196Y CTC, (c) W196A ox , and (d) W196A CTC wi h CHCHD4. The uppe plo s show he he mog ams ( he mal powe as a unc ion o ime), whe eas he lowe plo s show he binding iso he m (no malized hea s as a unc ion o he CHCHD4/hAIF mola a io). Measu emen s we e ca ied ou in 50 mM po assium phospha e, pH 7.4, a 25 ° C. The CTC o ms we e ob ained by p emixing hAIF Δ1-101ox and NADH a a 1 : 100 a io. The binding pa ame e s we e es ima ed h ough nonlinea leas -squa es eg ession applying a single-ligand binding model (con inuous lines in binding iso he ms). 16 Oxida i e Medicine and Cellula Longe i y ee ene gy o hAIF binding in bina y complexes wi h CHCHD4, CypA, and DNA. Whe eas, i s eplacemen has in gene al a nega i e impac on he en halpic binding con i- bu ion, while imp o es he en opic one (wi h he only excep ion o W196A CTC:CHCHD4 complex). The e o e, W196 con ibu es o s abilize he con o ma ion o he in e - ac ion su aces o hAIF wi h CHCHD4, CypA, and DNA. 3.7. W196 Con ibu es o Con ol he hAIF Con o ma ional Landscape o Adap o I s Physiological Roles. AIF is a moon- ligh p o ein wi h unc ions in he mi ochond ia, cy osol, and nucleus, whe e i appea s o beha e as a edox senso o NAD(H/ + ) cellula le els [9–11]. The cellula edox s a e (NAD + /NADH a io) may modula e he AIF con o ma ional landscape ega ding bo h o e all p o ein con o ma ion and qua e na y o ganiza ion, which in u n seems o be c i ical o es ablishing i s biomolecula in e ac ion ne wo k. The egula o y C-loop in AIF is a p edic ed in e nally diso de ed egion ha ends o adop an o ganized con o ma ion in he p o ein oxidized s a e (Figu e 1, Figu e S8 and S9), bu ha is eleased upon NADH-dependen p o ein educ ion, CTC o ma ion, and p o ein dime iza ion. The s uc u al p ope ies o W196A ox sugges ed ha he W196 side chain and he β-hai pin coupling o he C-loop a e key o modula e he s uc u al ansi ion o hAIF in a cellula con ex . In heal hy mi ochond ia, hAIF is p esen in a monome - dime equilib ium— egula ed by he cy oplasmic NADH pools— ha modula es i s pa icipa ion in espi a o y com- plex assembly by physical in e ac ions wi h CHCHD4 [10, 12–14]. This AIF swi ching may be c i ical o main aining mi ochond ial homeos asis along changes in NAD + /NADH a ios in esponse o die , diseases such as neu odegene a i e diso de s, and o he p ocesses associa ed o NAD- consuming enzymes—pa icula ly PARP-1 whose ac i i y is inc eased du ing aging due o DNA damage accumula- ion—[37, 38]. In esponse o NAD + deple ion by hype s im- ula ion o PARP-1, hAIF is eleased om he mi ochond ia o he cy osol, allowing i s ansloca ion o he nucleus and p omo ing pa hana os cell dea h. PARP-1 binding o AIF has been p o ed o media e i s elease om IM by likely inducing con o ma ional changes in he p o ein [39]. Cu i- ously, he exp ession le els o AIF we e ound o be g adually dec eased du ing de elopmen and g ow h in spi al ganglion neu ons in ol ed in audi o y neu opa hy spec um diso de , a disease caused by poin mu a ions in hAIF, while inc eased in he aging- ela ed cell dys unc ions whe e he hAIF ole as apop osis induce migh be mo e impo an [40]. To in es i- ga e he diffe en po en ial oles o AIF du ing de elopmen and aging and hei egula o y mechanisms, u u e s udies will be equi ed. In he las yea s, a significan numbe o a e mi ochon- d ial diseases caused by mo e han 20 poin mu a ions in he AIFM1 gene ha e been iden ified. Some mu a ions in he cell dea h domain gi e ise o pheno ypes wi h p og es- si e diso de s om childhood, as he Cowchock synd ome. On hei side, mu a ions affec ing he hAIF educ ase p ope - ies dec ease he con en o espi a o y complexes and p o- duce cell espi a ion deficiencies, while some o hem p e en in addi ion he co ec olding o he p o ein by dec easing i s con o ma ional s abili y. In his la e case, he sea ch o molecula chape ones ep esen s an al e na i e he apeu ic s a egy ye poo ly explo ed [7, 31, 41–44]. These mu a ions p oduce se ious mi ochond ial encephalopa hies, in many cases wi h se e e p ocesses o neu odegene a ion and ea ly dea h. No iceably, all cha ac e ized pa hogenic hAIF mu an s show a subs an ially dec ease in CTC li e ime, sugges ing ha i s s abili y is c ucial o mi ochond ial homeos asis and human heal h [18, 45]. Fu he molecula and cellula s udies will be equi ed o de e mine he impac o hese pa hogenic mu a ions on he hAIF in acellula p o- cessing and in e ac ion wi h i s physiological pa ne s, as well as hei link o hei mul iple clinical neu odegene a i e pheno ypes. 4. Conclusions This epo p o ides insigh s in o he ole in hAIF o W196 and β-hai pin mo i in he molecula basis o i s cellula ac i i ies. Ou mu a ional s udy shows ha , con a y o he pa hogenic ΔR201 mu a ion—ano he esidue loca ed in he β-hai pin and in ol ed in he in e ac ion wi h he egula- o y C-loop—, changes a W196 esidue ha dly impac he o e all con o ma ional olding o hAIF in oxidized and NADH- educed s a es. None heless, W196 is key o s abilize β-hai pin mo i con o ma ion by con ac s ha a e subs an- ially diminished and impai ed in all cha ac e ized W196 a ian s. Mo eo e , he W196 and he β-hai pin mo i con- o ma ion s ongly modula e he edox-linked monome - dime s uc u al ansi ion in hAIF. The size and a oma ici y o he side chain o W196 is key o (i) main ain he p ope β- hai pin posi ion ha s abilizes and e ains he egula o y C- loop in he p o ein sco e o oxidized hAIF, a o ing p o ein compac ness and s abili y; (ii) configu e he NADH ac i e si e making hAIF inefficien o NADH oxida ion and igge he C-loop elease o he sol en in he educed s a e: c i ical ac o s o CTC s abili y and mi ochond ial homeos asis; and (iii) define he in e ac ion su aces wi h CHCHD4, CypA, and DNA, by modula ing he en halpic and en opic con i- bu ions o he ee ene gy o binding. These ea u es con ib- u e o modula e hAIF monome -dime equilib ium in a cellula con ex , which migh be ele an o i s p ope unc- ion as a edox senso o NAD(H/ + ) le els and o i s in e ac- ion ne wo k. Abb e ia ions app MW: Appa en molecula weigh BS 3 : Homobi unc ional-bis[sul osuccinimidyl]- sube a e CHCHD4: Coiled-coil-helix-coiled-coil-helix domain con- aining 4 CTC: Cha ge ans e complex CD: Ci cula dich oism CypA: Cyclophilin A DCPIP: Dichlo ophenolindophenol FAD: Fla in adenine dinucleo ide hAIF: Human apop osis-inducing ac o 17Oxida i e Medicine and Cellula Longe i y hAIF ox : Oxidized hAIF hAIF d : Reduced hAIF hAIF ph d : Pho o educed hAIF IMS: In e memb ane space NADH: Reduced nico inamide adenine dinucleo ide NAD + : Oxidized nico inamide adenine dinucleo ide OXPHOS: Oxida i e phospho yla ion PARP-1: Poly(ADP- ibose) polyme ase-1 ROS: Reac i e oxygen species SAS: Sol en accessible su ace. Da a A ailabili y All da a a e con ained wi hin he manusc ip and he supple- men a y ma e ials. Con lic s o In e es The au ho s decla e ha hey ha e no conflic s o in e es . Acknowledgmen s This wo k was suppo ed by he Spanish Minis y o Economy, Indus y and Compe i i eness-S a e Resea ch Agency (MINECO, BIO2016-75183-P AEI/FEDER), Spanish Minis y o Science and Inno a ion-S a e Resea ch Agency (MICINN) (G an PID2019-103901GB-I00), and he Gobie no de A agón-FEDER [G upo de Re e encia Biología Es uc u al (E35_20R)]. A.V-C. hanks ARAID o financial suppo . Supplemen a y Ma e ials The file con ains he ollowing: (i) he p o ocol o p oduc- ion and pu ifica ion o p o eins and o MD simula ions and (ii) Figu es S1-S9. (Supplemen a y Ma e ials) Re e ences [1] S. A. Susin, H. K. Lo enzo, N. Zamzami e al., “Molecula cha - ac e iza ion o mi ochond ial apop osis-inducing ac o ,” Na u e, ol. 397, no. 6718, pp. 441–446, 1999. [2] H. K. Lo enzo, S. A. Susin, J. Penninge , and G. K oeme , “Apop osis inducing ac o (AIF): a phylogene ically old, caspase-independen effec o o cell dea h,”Cell Dea h and Diffe en ia ion, ol. 6, no. 6, pp. 516–524, 1999. [3] L. Dela allée, N. 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