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1 Supplementary Information Following evolutionary paths to high affinity an seletivity protein-protein interations Kalia Bernath Levin, Orly Dym, Shira Albek, Shlomo Magassi, Anthony H. Keeble, Colin Kleanthous an Dan S. Tawfik

2 a Im9 R5-4 R5-10 R5-11 R8-1 R8-6 R8-7 R8-4 R8-8 R12-2 R12-4 R12-13 Im7 b Supplementary Figure 1 Sequene alignment of Immunity proteins an oliins. (a) Amino-ai sequene alignment of the two wil types (Im9 an Im7) an nevim7 variants. (b) Amino-ai sequene alignment of ColE7 an ColE9 C-terminal region.

a b His-ColE7 H103A sr5-4 Im9 sr5-10 Im9

with one of Roun 5 variants ((a) sr5-4, (b)

with Ni-NTA resin, then loae onto a syringe

omplexes (his-e7-im protein) were elute with

SDS-PAGE analysis reveale the ratio between

The evolve variants were foun to bin ColE7

iniate by the isplaement of wil-type Im9,

3 a b His-ColE7 H103A sr5-4 Im9 sr5-10 Im9 sr5-11 Im9 1:1 2:1 4:1 8:1 1:1 2:1 4:1 8:1 1:1 2:1 4:1 8:1 Supplementary Figure 2 Competition between Roun 5 variants an Im9 for bining to ColE7. Equimolar onentration (3µM) of his-cole7 with one of Roun 5 variants ((a) sr5-4, (b) sr5-10, an () sr5-11, eah arrying a strep-tag), an wil-type Im9. ColE7, the evolve variant, an wil-type Im9, an were mixe at varying molar ratios of 1:1:1, 1:1:2, 1:1:4 an 1:1:8, respetively, (in 50mM MOPS ph 7, 50 mm NaCl buffer). The proteins were equilibrate overnight at room temperature. The mix was further inubate for two hours with Ni-NTA resin, then loae onto a syringe olumn, rinse one with buffer, an the omplexes (his-e7-im protein) were elute with buffer plus 300mM imiazole. SDS-PAGE analysis reveale the ratio between the evolve Im9 variant an wil-type Im9. The evolve variants were foun to bin ColE7 with higher affinity ompare to Im9, as iniate by the isplaement of wil-type Im9, even when the latter was present at higher onentration. Variants R5-10 an R5-10 exhibit higher affinity than variant R5-4, sine at a ratio of 1:8 with wil-type Im9, they were able to isplae Im9.

4 Arg530 Tyr54 Lys528 Tyr/Trp55 Phe541 Phe513 Supplementary Figure 3 The Tyr55Trp mutation in the onserve hot spot. The figure shows an overlay of the two nevim7 variants R12-2 (green, R12-2 an R12-13 in orange). Tyr55 in R12-2 is well aligne to the 5-membere inole ring, both are able to sustain the hyrophobi ore an reate a hyrogen bon to Lys528 bakbone (2.35Å an 2.79Å in an 12-2 respetively). However, the aitional bulkiness of the Trp may ontribute in expaning its hyrophobi interations to Phe541 an Phe513 (istanes in the range of 4Å-4.7Å) also leaing to the small shift in the alkyl hain of Arg530. The overall strutures of the two variants align very well also in their boun onfiguration.

a b Glu/Gly41 Glu41 Glu41 Thr531 Lys97 Lys528 e

The seletive inhibitors evolve by aquiring a

The similarity in struture an ColE7 bining

evolve variants (resiues 125 are missing for

5 a b Glu/Gly41 Glu41 Glu41 Thr531 Lys97 Lys528 e f Gly41 Val42 Thr531 Thr531 Supplementary Figure 4 The orthogonal aquisition of seletivity. The seletive inhibitors evolve by aquiring a single mutation at resiue 41. The similarity in struture an ColE7 bining onfiguration show that seletivity ourre inepenently from ColE7 inhibition. (a) Overall strutural alignment of the two evolve variants (resiues 125 are missing for larity): variant R12-2 (in green) that arries the seletivity Glu41Gly mutation an oes not ross-reat with ColE9; an R12-13 that retains Glu41, an

6 inhibits ColE9 to a measurable egree (orange). The piture is a result of whole omplex alignment. Resiues 54 an 55 are shown in stiks as referene. (b) A zoom in for the region of resiue 41. Resiue 41 sie-hain is highlighte in stiks, whereas only the bakbone of neighboring resiues are showen. () The boun Im9 (magenta an ColE9 (green). Glu41 makes a salt brige to ColE9 s Lys97 (3.18Å). () The boun variant R12-13 (yan) an ColE7 (green). The losest ColE7 resiues ontating R12-13 s Glu41 are Thr531 (3.37Å) an Lys528 (8.85Å). (e) The boun variant R12-2 (blue) an ColE7 (green).the losest ColE7 resiues to R12-2 s Gly41 are Thr531 (9.48Å). (f) The boun Im7 (re) an ColE7 (green). The losest ColE7 resiue to Im7 is Thr531 (6.67Å).

7 a E7_sR8-1 b E7_sR8-8 E7_sR12-4 Supplementary Figure 5 Representative SPR sensograms. Bining of variants to hips oate with ColE7was etete at various immunity protein onentrations: variant sr8-1 (a, 300, 500, 600nM), variant sr8-8 (b, 100, 200, 400nM), an variant sr12-4 (, 100, 200, 300nM). Dissoiation urves fitte to a single exponential (inset).

8 Denaturation urve Im9 R5-4 R5-10 R8-1 R8-7 R8-4 R12-2 Im7 + a Urea onentration (M) b Fration fole Fration fole Fration fole Urea onentration (M) Urea onentration (M) Supplementary Figure 6 Urea enaturation urves. Unfoling of immunity proteins was followe by measuring their fluoresene at 355nm in the presene of variousonentrations of urea 4. (a) The wil-type Im9 is muh more stable than wiltype Im7, whereas the variants stability lay between them, starting from high stability of Roun 5 variants to low in variant of Roun 12. Data were fitte to a two-state transition moel as esribe 4 an the resulting D 50 an m values are provie in Supplementary Table 1. (b) The erease in stability is most apparent in two of the intermeiate variants that aumulate loop mutations: R5-11 (b) an R8-6 (). These variants (an variant R8-

9 1, that showe the lowest m value; panel a o not arry the stabilizing mutation V37I, an seem to unfol non-ooperatively. The loss of stability is also manifeste in low expression levels an onsequently in the low in-vivo inhibition potenies of these variants towars both ColE9 an ColE7 (Supplementary Table 1). Experimental etails: 500µM of the strep-tag proteins were resuspene in buffer (50mM phosphate buffer ph 7, 2mM DTT, 1mM EDTA) an mixe 1:10 with inreasing onentration of urea in the same buffer. Following two hours inubation at room temperature, fluoresene was measure at room temperature in a Cary elipse (Varian) fluoresene spetrophotometer using exitation at 280nm; an emission at 355 nm measure at room temperature.

His-ColE7 H103A a His-ColE7 H103A b sr12-4 Im7 Time: 22min 30min 45min 2hrs 20hrs % replae: 24 70 64 72.6 71 sr12-13 Im7 Time: 22min 30min 45min 2hrs % replae: 63 91.5 98.3 99.

Pre-mae omplexes of ColE7 an Im variant arrying a strep-tag (sr12-4, a, or sr12-13, b, 1µM, in 50mM MOPS ph 7, 200mM NaCl) were hase with a ten-fol exess of wil-type Im7 without a tag (10 µm).

10 His-ColE7 H103A a His-ColE7 H103A b sr12-4 Im7 Time: 22min 30min 45min 2hrs 20hrs % replae: sr12-13 Im7 Time: 22min 30min 45min 2hrs % replae: Supplementary Figure 7 Dissoiation kinetis by Im protein exhange. Slow issoiation kinetis were measure by a hase experiment. Pre-mae omplexes of ColE7 an Im variant arrying a strep-tag (sr12-4, a, or sr12-13, b, 1µM, in 50mM MOPS ph 7, 200mM NaCl) were hase with a ten-fol exess of wil-type Im7 without a tag (10 µm). At ifferent time points, Ni-NTA resin (Qiagen) was ae an the mix was inubate for 15 minutes while shaking. The unboun supernatant was rapily ispose by loaing the mix onto a syringe-olumn. The resin was rinse with the same buffer an the boun omplex was elute with buffer plus 300mM imiazole. The eluants were onentrate with a Nanosep 3K (Pall orporation) an analyse by 15% SDS- PAGE. The ratio between the protein bans orresponing to the strep-tagge Im-variant (MW= 11.3kDa) an wil-type Im7 (MW= 9.9kDa) were measure by ensitometry using the GeneQuant software. In aorane with the SPR results, the t 1/2 for E7 sr12-4 issoiation was between 22 to 30 mins (k off SPR= s -1, t 1/2 =14.4 mins), an the t 1/2 for E7 sr12-13 issoiation was 20 mins (k off SPR= s -1, t 1/2 =8.9 mins).

11 Supplementary Table 1. Inhibition poteny, bining, an stability of Im variants Variant a Stability Urea enatura tion m kal/m ol/m urea D 50 (M) 1mM IPTG In vivo protetion ColE7 (M) b 0.05 mm IPTG In vivo inhibition poteny an affinity ColE7 ColE9 Bining parameters Stoppe-flow In vivo protetion /SPR ColE9 (M) b Basal k on ( 10 6 M -1 s -1 ) k off (s -1 ) Wiltype Im e 5.34 Im9 V34D R K (M) 1mM IPTG 0.05 mm IPTG Basal Bining parameters Stoppe-flow /SPR k on ( 10 6 k off (s -1 ) K (M) M -1 s -1 ) >10-4 >10-4 > e >10-4 >10-4 > n. f >10-4 >10-4 >10-4 f R <10-8f >10-4 >10-4 >10-4 f i i R <10-8f >10-4 > f 10-8 R n. > R8-6 R8-6 E41G i i n. >10-4 > n R >> R8-7 E41G (±24) n. -4 > >10-4 >10-4 >> >10-4 > R8-8 h h >>10-4 >> R8-8 E41G R >> >10-4 >10-4 >10-4 >>10-4 >> > R >>10-4 >> R12-4 h h >>10-4 >>10-4 >>10-4 R12-13 h h >>10-4 >>10-4 >>10-4 Wiltype Im >10-4 >10-4 >10-4 ~ g > g > g >10-4 >10-4 > >>10-4 >>10-4 >> e n. ~ ~ g ~ g ~ g n. n. 10-4e a The numbering of the nevim7 variants (e.g. R8-7) iniates the roun of mutationseletion from whih it was isolate (e.g. Roun 8), an the sequential number of the

12 variant in the series analyze (e.g. the 7 th variant). The omplete sequenes are available in Supplementary Fig. 1. b The in vivo protetion levels were etermine with E. oli ells expressing the enote Im variant, treate with inreasing onentrations of either ColE7, or ColE9 omplex (10-4 to M). The table iniates the maximal onentration of ColE uner whih E. oli growth remaine unaffete. The test was performe at ifferent Im protein expression levels: basal in the absene of the IPTG inution; low inution levels (0.05M IPTG), an full inution (1M IPTG) 1., Bining parameters were etermine by stoppe-flow () 2, or SPR () 3 (see Supplementary Table 2 for ata an error ranges, whih were below 10%). The two measurements gave essentially the same k off values with ColE9, but the values measure by SPR for issoiation from ColE7 eviate onsistently by a fator of ~3. The k off values for Roun 12 variants were also onfirme by exhange an ompetition experiments (Supplementary Fig. 7). e Bining parameters from 1. f The issoiation rates for Roun 5 were off-range for SPR: issoiation from ColE7 was too fast, an from ColE9 too slow. Their higher affinity towars ColE7 relative to Im9 was onfirme by ompetition experiments. These iniate that variants R5-10 an R5-11 exhibit > 10-fol higher affinity than Im9, sine at a ratio of 1:8 with wil-type Im9, they were still able to isplae Im9 (Supplementary Fig. 2). g K values were eue assuming the average k on vales are measure in the stoppe flow for Roun 8 variants, an k off values measure by SPR (as is for ColE9, an orrete for ColE7 where ~3-fol slower rates were measure by stoppe-flow. h These variants arry the Trp55 mutation that prevente etetion of enaturation by fluoresene emission. i Denaturation appears to be non-ooperative an oul not be fitte to a sigmoial moel (Supplementary Fig. 5).

13 Supplementary Table 2. Surfae Plasmon Resonane (SPR) measurements ColE9 issoiation rates Averag Variant k off se k off se ColE7 issoiation rates Error range 10-3 k off se Averag k off se Error range 10-3 sr5-4 sr5-10 sr5-11 sr sr sr R8-7 * sr sr sr sr12-4 * sr The k off values etermine by SPR (BIAore) are a result of 2-3 inepenent measurements after reution of bakgroun signals (representative traes are given as Supplementary Fig. 6). Variants were mostly purifie with a sterp-tag (Novagen) labele with an s in front of the variant s annotation. However, variant R8-7 that was assaye with an without a tag iniate that the tag ha no effet on the bining parameters. Bakgroun signals were taken from either the wil-type sensograms (Im9 or Im7, whose k off is too slow for issoiation within the time sale of the experiment), or BSA in ases where in whih the wil-type an variant sensograms were not well synhronize uring the experiment s timesale (annotate with *). The variants onentration was between nm. Variants with issoiation rates beyon the measuring range of BIAore are marke as not etermine (). These rates were evaluate by other methos (Supplementary Fig. 2 & 7).

14 Referenes 1. Li, W. et al. Highly isriminating protein-protein interation speifiities in the ontext of a onserve bining energy hotspot. J Mol Biol 337, (2004). 2. Wallis, R. et al. Protein-protein interations in oliin E9 DNase-immunity protein omplexes. 2. Cognate an nonognate interations that span the millimolar to femtomolar affinity range. Biohemistry 34, (1995). 3. Kortemme, T. et al. Computational reesign of protein-protein interation speifiity. Nat Strut Mol Biol 11, (2004). 4. Ferguson, N., Capali, A.P., James, R., Kleanthous, C. & Rafor, S.E. Rapi foling with an without populate intermeiates in the homologous four-helix proteins Im7 an Im9. J Mol Biol 286, (1999).

Supplementary Materials for A universal data based method for reconstructing complex networks with binary-state dynamics

Supplementary Materials for A universal ata ase metho for reonstruting omplex networks with inary-state ynamis Jingwen Li, Zhesi Shen, Wen-Xu Wang, Celso Greogi, an Ying-Cheng Lai 1 Computation etails