The trigger factor chaperone from Thermotoga maritima - 3GU0.pdb

Fig. Fs1A C-terminus

PPIase domain

N-terminus

NP_000792 ------------------------------------------------------------!NP_228503 MEVKELERDKNRVVLEYVFGAEEIAQAEDKAVRYLNQRVEIPGFRKGRIPKNVLKMKLGE! !

!

NP_000792 ------------------------------------------------------------!

NP_228503 EFQEYTLDFLMDLIPDTLKDRKLILSPIVTERELKDVTARVVVEVHEEPEVRIGDISKIE! !!

NP_000792 -------------MGVQVETISPGDGRTFPKRG-----QTCVVHYTGMLEDGKKFDS---!NP_228503 VEKVDEEKVLEKYVERRIEDLRESHALLEPKEGPAEAGDLVRVNMEVYNEEGKKLTSREY!

: ::* : ... **.* : *: *:***: * !!

NP_000792 ----SRDRNKPF-KFMLGKQ-----EVIRGWEEG-----VAQMSVGQRAKLTISPDYAYG!

NP_228503 EYVISEDEDRPFVKDLVGKKKGDVVEIEREYEGKKYTYKLEVEEVYKRTLPEIGDELAKS! *.*.::** * ::**: *: * :* : .* :*: *. : * .!!

NP_000792 ATGHPG-------IIPPHATLVFDVELLKLE-----------------------------!NP_228503 VNNEFETLEQLKESLKKEGKEIYDVEMKESMREQLLEKLPEIVEIEISDRTLEILVNEAI!

.... : ... ::***: : !!

NP_000792 ------------------------------------------------------------!

NP_228503 NRLKREGRYEQIVSSYESEEKFREELKERILDDIKRDRVIEVLAQEKGISVNDEELEKEA! !

!

NP_000792 ------------------------------------------------------------!NP_228503 EELAPFWGISPDRAKSLVKARQDLREELRWAILKRKVLDLLLQEVKVKVVEPKGEGDDSE!

!!

NP_000792 -----!NP_228503 GKEDN!

Fig. Fs1B Sequence alignment of hFKBP12a with trigger factor from Thermotoga maritima MSB8

(yellow - structural !PPIase"-domain" as shown in the above X-ray structure)

Figure Fs1. (A) X-ray structure of the trigger factor from Thermotoga maritiama

(3GU0.pdb); residues from 1 to 405; resolution at 3.5 Å [Rs1]. PPIase domain is in a

gray circle; (B) Sequence alignment of the above mentioned protein (NP_228503) with

the hFKBP12a (NP_000792). Sequence similarity score ID is about 7%, which indicates

for a total disparity of their PPIase domains. The PPIase domain of the trigger factor

(Thermotoga maritiama) is smaller (about 75 AAs) as compared to 107 AAs of the

hFKBP12a; the sequence fragment in yellow corresponds to the PPIase domain as

indicated in the structure shown above. There is a distant sequence-structure similarity

between the trigger factors and the FKBPs.

Rs1. Martinez-Hackert E, Hendrickson WA (2009) Promiscuous substrate recognition

in folding and assembly activities of the trigger factor chaperone. Cell 138:923-934

N-terminus

C-terminus

EGCG EGCG

Figure Fs2A

Structural and functional insights of directly targeting Pin1 by EpiGalloCatechin-3-Gallate (EGCG)

C-terminus

N-terminus

Fig. Fs2B Crystal structure of human parvulin 14 (3UI5.pdb)

!

3UI5_A -----------------------------------GPMGSN------------AVKVRHI!

hPin1 MADEEKLPPGWEKRMSRSSGRVYYFNHITNASQWERPSGNSSSGGKNGQGEPARVRCSHL!

hFKBP12a ---------------------------MGVQVETISPGDGR----------TFPKRGQTC!

* .. : !

!

3UI5_A LCEKHG---------------------KIMEAMEKLKSG-MRFNEVAAQYSE-DKARQGG!

hPin1 LVKHSQSRRPSSWRQEKITRTKEEALELINGYIQKIKSGEEDFESLASQFSDCSSAKARG!

hFKBP12a VVHYTG----------------------MLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWE!

: . : :*:.*. : . .. . : !

!

3UI5_A D-LGWMTRGSMVGPFQEAAFALPVSGMDKPVFTDPPVKTKFGYHIIMVEGRK!

hPin1v D-LGAFSRGQMQKPFEDASFALRTGEMSGPVFTDS------GIHIILRTE--!

hFKBP12a EGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHAT--LVFDVELLKLE---!

: :. :: *. .. :* . : ... . .:: !

Fig. Fs2C Sequence alignment of human Parvulin14 with human Pin1 and hFKBP12a

Figure Fs2. (A) X-ray structure of the human Pin1 (green ribbon, hPin1) bound to

Epigallocatechin-3-gallate (red sphere; [(2R,3R)-5,7-dihydroxy-2-(3,4,5-

trihydroxyphenyl)chroman-3-yl] 3,4,5-trihydroxybenzoate); in blue spheres are shown 2-{2-

[2-(2-{2-[2-(2-ethoxy-ethoxy)-ethoxy]-ethoxy}-hetnamethoxy)-ethoxy]-ethoxy}-ethanol;

3OOG.pdb; resolution 1.89 Å [Rs2]; (B) Crystal structure of human Parvulin 14 (green

ribbon, hPar14) 3UI5.pdb; resolution 0.8 Å [Rs3]; (C) Sequence alignment of human

Parvulin 14 (hPar14) shown above with hPin1 and hFKBP12a. Sequence similarity score ID

for the pair hFKBP12a/hPin1 is about 23%, whereas there is no sequence similarity between

hFKBP12/hPar14 (ID = 4%) and hPar14/hPin1 (ID = 8%). The Pin1s and parvulins have no

sequence/structure similarity with the FKBPs.

Rs2. Urusova DV, Shim JH, Kim DJ, Jung SK, Zykova TA, Carper A, Bode AM, Dong Z (2011) Epigallocatechin-gallate suppresses tumorigenesis by directly targeting Pin1. Cancer Prev Res (Phila) 4:1366-1377 Rs3. Mueller JW, Link NM, Matena A, Hoppstock L, Ruppel A, Bayer P, Blankenfeldt W (2011) Crystallographic proof for an extended hydrogen-bonding network in small prolyl isomerases. J Am Chem Soc 133:20096-20099

Figure Fs3. The MSA25 of the human FKBDs. 1 hFKBP12a 1-MGVQVETISPGD-GRTFPKRGQTCVVHYTGMLED-GKKFDS----SRDRNK---PFKFML 2 hFKBP12b 1-MGVEIETISPGD-GRTFPKKGQTCVVHYTGMLQN-GKKFDS----SRDRNK---PFKFRI 3 hFKBP12c 1-MGVHVETISPGD-WRTFPKRSQTCVMHYTGMLED-GKKFDS----SRDRNK---PFKFML 4 hFKBP25 109-PKYTKSVLKKGD-KTNFPKKGDVVHCWYTGTLQD-GTVFDTNIQTSAKKKKNAKPLSFKV 5 hFKBP133 180-AVLSQDLIVADG---PAVEVGDSLEVAYTGWLFQ-NHVLGQVFDSTANKDK---LLRLKL 6 hFKBP15/p 30-KLQIGVKKRVDH-CPIKSRKGDVLHMHYTGKLE--DGTEFDS---SLPQN---QPFVFSL 7 hFKBP22/p 37-TLQVETLVEPPEPCAEPAAFGDTLHIHYTGSLV--DGRIIDT---SLTR----DPLVIEL 8 hFKBP24/p 29-EVKIEVLQKPFI-CHRKTKGGDLMLVHYEGYLEK-DGSLFHS---THKHNNG-QPIWFTL 9 hFKBP25_ER 36-EVKIEVLHRPEN-CSKTSKKGDLLNAHYDGYLAK-DGSKFYC---SRTQNEG-HPKWFVL 10 hFKBP63_1 35-ELQIERRFVPDE-CPRTVRSGDFVRYHYVGTFP--DGQKFDS---SYDRD---STFNVFV 11 hFKBP63_2 147-QVQIHTYFKPPS-CPRTIQVSDFVRYHYNGTFL--DGTLFDS---SHNRM---KTYDTYV 12 hFKBP63_3 259-SISIENKVVPEN-CERISQSGDFLRYHYNGTLL--DGTLFDS---SYSRN---RTFDTYI 13 hFKBP63_4 370-SISITSHYKPPD-CSVLSKKGDYLKYHYNASLL--DGTLLDS---TWNLG---KTYNIVL 14 hFKBP64_1 43-DVVIERYHIPRA-CPREVQMGDFVRYHYNGTFE--DGKKFDS---SYDRN---TLVAIVV 15 hFKBP64_2 155-TVQVSTLLRPPH-CPRMVQDGDFVRYHYNGTLL--DGTFFDT---SYSKG---GTYDTYV 16 hFKBP64_3 267-AVQLETLELPPG-CVRRAGAGDFMRYHYNGSLM--DGTLFDS---SYSRN---HTYNTYI 17 hFKBP64_4 379-VVEIRTLSRPSETCNETTKLGDFVRYHYNCSLL--DGTQLFT---SHDYG---APQEATL 18 hFKBP52_1 31-EGVLKVIKREGT-GTEMPMIGDRVFVHYTGWLLD-GTKFDS----SLDRKD---KFSFDL 19 hFKBP52_2 148-GGIIRRIQTRGE-GYAKPNEGAIVEVALEG--YY-KDKLFD------QRE-----LRFEI 20 hFKBP51_1 31-RGVLKIVKRVGN-GEETPMIGDKVYVHYKGKLSN-GKKFDS----SHDRNE---PFVFSL 21 hFKBP51_2 146-GGIIRRTKRKGE-GYSNPNEGATVEIHLEG--RC-GGRMFD------CRD-----VAFTV 22 hFKBP38 100-GLLRKKTLVPGPPGSSRPVKGQVVTVHLQTSLEN-GTRVQE------EPE-----LVFTL 23 hFKBP36 31-RGVLKDVIREGA-G-DLVAPDASVLVKYSGYLEH-MDRPFDS---NYFRKT---PRLMKL 24 hFKBP37 11-DGIQKRVIQEGRGELPDFQDGTKATFHYRTLHSDDEGTVLDD---SRARGK---PMELII 25 hFKBPL37 10-EGVKKTILHGGTGELPNFITGSRVIFHFRTMKCDEERTVIDD---SRQVGQ---PMHIII . : 1 hFKBP12a GKQ---EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHP-GIIPPHATLVFDVELLKLE-108* 2 hFKBP12b GKQ---EVIKGFEEGAAQMSLGQRAKLTCTPDVAYGATGHP-GVIPPNATLIFDVELLNLE-108* 3 hFKBP12c GKQ---EVIRGWEEGVVQMSVGQRAKLTISPDYAYGATGHP-GIIPPHATLVFDVELLKLE-108* 4 hFKBP25 GVG---KVIRGWDEALLTMSKGEKARLEIEPEWAYGKKGQPDAKIPPNAKLTFEVELVDID-224* 5 hFKBP133 GSG---KVIKGWEDGMLGMKKGGKRLLIVPPACAVGSEGVIGWTQATDSILVFEVEVRRVK-290 6 hFKBP15/p GTG---QVIKGWDQGLLGMCEGEKRKLVIPSELGYGERG-APPKIPGGATLVFEVELLKIE-137 7 hFKBP22/p GQK---QVIPGLEQSLLDMCVGEKRRAIIPSHLAYGKRG-FPPSVPADAVVQYDVELIALI-144 8 hFKBP24/p GIL---EALKGWDQGLKGMCVGEKRKLIIPPALGYGKEG--KGKIPPESTLIFNIDLLEIR-135 9 hFKBP25_ER GVG---QVIKGLDIAMTDMCPGEKRKVVIPPSFAYGKEGYAEGKIPPDATLIFEIELYAVT-145 10 hFKBP63_1 GKG---QLITGMDQALVGMCVNERRFVKIPPKLAYGNEG-VSGVIPPNSVLHFDVLLMDIW-145 11 hFKBP63_2 GIG---WLIPGMDKGLLGMCVGEKRIITIPPFLAYGEDG-DGKDIPGQASLVFDVALLDLH-256 12 HFKBP63_3 GQG---YVIPGMDEGLLGVCIGEKRRIVVPPHLGYGEEG-RGN-IPGSAVLVFDIHVIDFH-365 13 HFKBP63_4 GSG---QVVLGMDMGLREMCVGEKRTVIIPPHLGYGEAG-VDGEVPGSAVLVFDIELLELV-480 14 HFKBP64_1 GVG---RLITGMDRGLMGMCVNERRRLIVPPHLGYGSIG-LAGLIPPDATLYFDVVLLDVW-150 15 HFKBP64_2 GSG---WLIKGMDQGLLGMCPGERRKIIIPPFLAYGEKG-YGTVIPPQASLVFHVLLIDVH-264 16 hFKBP64_3 GQG---YIIPGMDQGLQGACMGERRRITIPPHLAYGENG-TGDKIPGSAVLIFNVHVIDFH-374 17 HFKBP64_4 GAN---KVIEGLDTGLQGMCVGERRQLIVPPHLAHGESG-ARG-VPGSAVLLFEVELVSRE-486 18 hFKBP52_1 GKG---EVIKAWDIAIATMKVGEVCHITCKPEYAYGSAGSP-PKIPPNATLVFEVELFEFK-138 19 hFKBP52_2 GEGENLDLPYGLERAIQRMEKGEHSIVYLKPSYAFGSVGKEKFQIPPNAELKYELHLKSFE-253 20 hFKBP51_1 GKG---QVIKAWDIGVATMKKGEICHLLCKPEYAYGSAGSL-PKIPSNATLFFEIELLDFK-138 21 hFKBP51_2 GEGEDHDIPIGIDKALEKMQREEQCILYLGPRYGFGEAGKPKFGIEPNAELIYEVTLKSFE-251 22 hFKBP38 G---DCDVIQALDLSVPLMDVGETAMVTADSKYCYGPQGSRSPYIPPHAALCLEVTLKTAV-205 23 hFKBP36 GED---ITLWGMELGLLSMRRGELARFLFKPNYAYGTLG-CPPLIPPNTTVLFEIELLDFV-138 24 hFKBP37 GKK---FKLPVWETIVCTMREG------------------LDALQQNPQPLIFHMEMLKVE-159 25 hFKBPL37 GNM---FKLEVWEILLTSMRVH------------------LDELQKEPQPLVFVIELLQVD-158 * : : : :

Probably, this sequence insert has !-helical stucture 24 hFKBP37 84-EIAQFLCDIKHVVLYPLVAKSLRNIAVGKDPLEGQRHCCGVAQMREHSSLGHAD-138 25 hFKBPL37 83-EVAEFWCDTIHTGVYPILSRSLRQMAQGKDPTEWHVHTCGLANMFAYHTLGYED-137 *:*:* ** *. :**::::***::* **** * : * **:*:* : :**: *

Figure Fs3. MSA of 25 FKBDs (MSA25) from the human FKBPs with indicated specific

sequence hallmarks, namely the –AYG-motif and alike are in red ink, the cysteine residues in

the FKBDs from the ER-anchored FKBPs are in yellow, whereas NLS signal in the nuclear

FKBP25 is in pink ink. The sequence motif, which may extend the FKBD in the FKBP-like

proteins (no. 24 and 25), matches to a TPR motif from the 1564 AAs-long TPR repeat protein

37 (NP_055454). Sequence indexes indicate the borders of the FKBDs while the (*) sign

means the C-terminus AA. The FKBD from hFKBP37 has the lowest sequence similarity

score with the hFKBP12A (ID = 13%) but it is about ID = 20% if a part of C-terminal

sequence is shifted as shown in this figure. The sequence in the gap has a good overlap with

one of the TPRs in the TTC37 protein [Rs4]. A recent NMR study on the FKBD from

hFKBP37 has suggested that its fold may substantially depart from the archetypal FKBP

structural template [Rs5]. The MSA25 and MSA500 were made with the ClustalX program

[24] on a Macintosh computer.

Rs4. Fabre A, Martinez-Vinson C, Roquelaure B, Missirian C, André N, Breton A, Lachaux

A, Odul E, Colomb V, Lemale J, Cézard JP, Goulet O, Sarles J, Levy N, Badens C (2011)

Novel mutations in TTC37 associated with tricho-hepato-enteric syndrome. Hum Mutat

32:277-281

Rs5. Linnert M, Haupt K, Lin YJ, Kissing S, Paschke AK, Fischer G, Weiwad M, Lucke C

(2012) NMR assignments of the FKBP-type PPIase domain of the human aryl-hydrocarbon

receptor-interacting protein (AIP). Biomol NMR Assign PMID: 22287093

0

0,5

1

1,5

2

2,5

3

3,5

4

3 12 21 30 39 48 57 66 75 84 93 102

MSA500-derived information entropy (Ie)

Info

rmation e

ntr

opy

Sequence index

Figure Fs4. (A) Information entropy graph (Ie) derived from the MSA500; if in a given

column the number of AAs is lower than 25%, it has an arbitrarily assigned negative artificial

sign for sake of graphic presentation. (B) A digital file for the above figure.

Figure Fs4B. Digital file for the graphics shown on Fig. Fs4A. 1

2

Information entropy for: MSA500 with hFKBP12 as reference 3

Sequence. The sign * indicates that Ie is below 1 and 4

implies a good conservation of the sequence position; 5

negative Ie artificially indicates that the sequence 6

position has a low occupancy. 7

8

Number of entries in the MSA = 500 9

Number of sequence positions in the MSA = 179 10

Number of positions in the leading sequence = 108 11

12

## Fill Info-Entropy Sequence 13

14

1 225 -0.250 M 15

2 320 1.768 G 16

3 432 1.969 V 17

4 437 3.496 Q 18

5 440 2.471 V 19

6 447 3.347 E 20

7 479 3.144 T 21

8 485 2.870 I 22

9 485 3.267 S 23

10 183 -0.250 P 24

11 214 -0.250 G 25

12 387 2.563 D 26

13 481 2.537 G 27

14 483 3.765 R 28

15 472 3.554 T 29

16 489 3.382 F 30

17 497 1.795 P 31

18 497 2.953 K 32

19 496 3.368 R 33

20 497 0.909 G * 34

21 497 2.456 Q 35

22 497 3.195 T 36

23 497 1.809 C 37

24 468 3.455 V 38

25 499 2.043 V 39

26 500 1.570 H 40

27 500 0.489 Y * 41

28 500 2.454 T 42

29 500 0.623 G * 43

30 494 2.731 M 44

31 500 0.935 L * 45

32 417 3.076 E 46

33 444 2.887 D 47

34 428 0.937 G * 48

35 432 2.428 K 49

36 284 2.264 K 50

37 486 0.912 F * 51

38 488 0.782 D * 1

39 494 1.194 S 2

40 493 1.391 S 3

41 457 2.549 R 4

42 492 2.732 D 5

43 494 1.874 R 6

44 492 2.081 N 7

45 446 3.282 K 8

46 495 1.369 P 9

47 496 1.455 F 10

48 499 3.250 K 11

49 500 1.168 F 12

50 499 3.124 M 13

51 500 1.700 L 14

52 500 0.321 G * 15

53 500 3.216 K 16

54 498 1.473 Q 17

55 494 2.736 E 18

56 499 0.804 V * 19

57 499 0.619 I * 20

58 500 2.050 R 21

59 500 0.854 G * 22

60 500 1.063 W 23

61 500 1.013 E 24

62 500 2.675 E 25

63 500 1.084 G 26

64 500 1.808 V 27

65 500 2.760 A 28

66 500 2.733 Q 29

67 500 0.585 M * 30

68 500 2.530 S 31

69 500 2.491 V 32

70 500 0.569 G * 33

71 500 1.472 Q 34

72 500 2.689 R 35

73 500 1.725 A 36

74 500 3.016 K 37

75 500 1.577 L 38

76 500 2.346 T 39

77 500 1.856 I 40

78 497 2.780 S 41

79 497 1.521 P 42

80 493 2.579 D 43

81 496 2.234 Y 44

82 498 1.102 A 45

83 499 0.452 Y * 46

84 500 0.349 G * 47

85 499 3.001 A 48

86 499 3.178 T 49

87 470 0.502 G * 50

88 489 3.368 H 51

89 490 1.894 P 52

90 451 2.298 G 1

91 404 3.159 I 2

92 495 0.615 I * 3

93 496 0.436 P * 4

94 484 1.728 P 5

95 485 1.905 H 6

96 428 1.471 A 7

97 489 1.765 T 8

98 488 0.719 L * 9

99 489 2.730 V 10

100 489 0.578 F * 11

101 483 1.555 D 12

102 483 1.150 V 13

103 480 1.034 E 14

104 478 0.701 L * 15

105 477 2.057 L 16

106 475 3.069 K 17

107 472 2.736 L 18

108 430 3.319 E 19

20

Figure Fs5 (A+B)

(A) C-terminal domain of the protein C56c10.10, a putative peptidylprolyl

isomerase from Caenorhabditis elegans - 3RKV.pdb!

TPR-I

TPR-II

TPR-III

C-terminus

Sequence alignment of the FKBD in AIP from C.elegans (Q18888) with hFKBP12a

B

Figure Fs5. (A) C-terminal domain of protein C56c10.10, a putative peptidylprolyl isomerase

from Caenorhabditis elegans - residues 184-362 - 3RKV.pdb; Resolution 2.41 Å; [Rs6].

TPR-I (red); TPR-II (violet); TPR-III (red); and C-terminal !-helix (orange); (B) Alignment

of the entire sequence of the AIP protein (Q18888) with the hFKBP12a; the sequence of

3RKV.pdb corresponds to the C-terminal domain of Q18888.

Rs6. Osipiuk J, Tesar C, Gu M, VAN Oosten-Hawle P, Morimoto RI, Joachimiak A (2011) 3RKV.pdb submitted to the RCSB by Midwest Center for Structural Genomics (MCSG)

FKBP

TPR-I

TPR-II

TPR-III

Figure Fs6 3RZE.pdb 2IF4.pdb

TM-V

TM-VI

TM-III

TM-II

TM-I

TM-IV

TM-VII

Figure Fs6. Two X-ray structures of: GPCR - (3RZE.pdb) entitled ‘Structure of the human

histamine H1 receptor in complex with doxepin’, at left panel; resolution 3.10 Å [Rs7]; the

TPR-containing AtFKBP42 (right panel) [53]. These two helix bundles have some structural

similarity, namely the seven-helix bundle (GPCR) is composed of hydrophobic helices, which

fit tightly with each other forming an opening at the top (extracellular part of the GPCR) to

which may enter different ligands. TPRs (I-III) in the second structure are made of

amphipathic helices. If we use the following two hypotheses on how to make conversion of

TPR motif bundle into hydrophobic helix bundle: 1) if each of the TPR motifs becomes

straight, then we would have a three helix bundle. In a GPRC, its components should increase

their hydrophobicity levels; 2) each of the helices in the TPR bundle increases its length and

hydrophobicity and in such a fashion that a seven helix bundle is formed, with the seventh

helix being the C-terminus helix as shown in Fig. 7B [62]. Even at their current forms, both of

the !-helical bundles shown here have similar intra-molecular interaction patterns [48].

Moreover, the following arguments could support the hypothesis that some of the TPR

motifs-containing FKBPs could be associated to various hydrophobic moieties in the cell,

namely 1° conformational polymorphism of the TRP motifs that has been observed in the two

crystallographic forms of bovine cyclophilin-40 [52] might be a general in vivo operational

property of the TPR motifs in some of the FKBPs; and 2° the C-terminal TM segment (HI >

55%) of hFKBP38 is embedded in the membrane, its two hydrophobic TPR motifs TPR-II

and TPR-III could interact with some membrane proteins due to their high HIs and basic pIs,

whereas its hydrophobic PPIase domain could target accessible X-Pro epitopes in some target

proteins.

Rs7. Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, Abagyan R,

Cherezov V, Liu W, Han GW, Kobayashi T, Stevens RC, Iwata S (2011) Structure of the

human histamine H1 receptor complex with doxepin. Nature 475:65-70

ARRE1

NFATc1

K192

L318

V678

W399

R267

Major groove

Minor groove

cJun

dA (-)

dT (+) cFos

Fig. Fs7

Figure Fs7. The X-ray structure (1S9K.pdb) of the ARRE1 double helix DNA bound to the

complex containing NFATc1 (violet), cFos (orange), and cJun (green) [76]. DNA sequence

starts from A-chain (+) 5’-TTTGAAAATATGTGTAATAG and B-chain (-) 5’-

ACTATTACATATTTTCAA. The DNA binding domain of NFATc1 starts at W399 (pale

green) and ends with V676 (cyan). The !-helices of the AP1 transcription factor (cFos +

cJun) are anti-parallel and bind at the major groove of the DNA fragment.

Figure Fs8. 3SYX.pdb (WH1 domain)

N-ter

C-ter

Figure Fs8. The X-ray structure of pleckstrin homology-like (PH) domain, also known as

WH1 domain, from the human sprouty-related EVH1 domain-containing protein

(3SYX.pdb); resolution 2.45 Å [Rs8].

Rs8. Vorobiev S, Su M, Seetharaman J, Sahdev S, Xiao R, Ciccosanti C, Shastry R, Everett JK, Nair R, Acton TB, Rost B, Montelione GT, Tong L, Hunt JF (2011) Crystal structure of the WH1 domain from human sprouty-related, EVH1 domain-containing protein. Northeast

Structural Genomics Consortium (NESG) - to be published; accessible from the Research Collaboratory for Structural Bioinformatics server (RCSB, http://www.rcsb.org)

Figure Fs9. Domain structure of calcineurin A.

Subunit ! (isoform 1) of Calcineurin A (521 AAS, NP_000935.1, GI:6715568, map

4q24, gene PPP3CA, Homo sapiens). N-terminus is in green box whereas in blue is

catalytic domain followed with calcineurin B binding domain (deep red) and

autoinhibitory domain (pink); in red are shown Zn and Fe binding sites, S in cyan

is potential phosphoserine. There are also Subunit ! (isoform 2) of calcineurin A

(511 AAs, NP_001124163.1 GI:194688147) and isoform 3 (469 AAs, NP_001124164.1 GI:

194688149).

1 MSEPKAIDPK LSTTDRVVKA VPFPPSHRLT AKEVFDNDGK PRVDILKAHL MKEGRLEESV

61 ALRIITEGAS ILRQEKNLLD IDAPVTVCGD IHGQFFDLMK LFEVGGSPAN TRYLFLGDYV

121 DRGYFSIECV LYLWALKILY PKTLFLLRGN HECRHLTEYF TFKQECKIKY SERVYDACMD

181 AFDCLPLAAL MNQQFLCVHG GLSPEINTLD DIRKLDRFKE PPAYGPMCDI LWSDPLEDFG

241 NEKTQEHFTH NTVRGCSYFY SYPAVCEFLQ HNNLLSILRA HEAQDAGYRM YRKSQTTGFP

301 SLITIFSAPN YLDVYNNKAA VLKYENNVMN IRQFNCSPHP YWLPNFMDVF TWSLPFVGEK

361 VTEMLVNVLN ICSDDELGSE EDGFDGATAA ARKEVIRNKI RAIGKMARVF SVLREESESV

421 LTLKGLTPTG MLPSGVLSGG KQTLQSATVE AIEADEAIKG FSPQHKITSF EEAKGLDRIN

481 ERMPPRRDAM PSDANLNSIN KALTSETNGT DSNGSNSSNI Q*

Subunit " (isoform B) of Calcineurin A (524 AAs, NP_066955, GI:11036640, map

10q22.2, gene PPP3CB, Homo sapiens). (Isoform A, 525 AAs, NP_001135825.1, GI:

215272352; isoform C, 515 AAs, NP_001135826.1, GI: 215272354)

1 MAAPEPARAA PPPPPPPPPP PGADRVVKAV PFPPTHRLTS EEVFDLDGIP RVDVLKNHLV

61 KEGRVDEEIA LRIINEGAAI LRREKTMIEV EAPITVCGDI HGQFFDLMKL FEVGGSPANT

121 RYLFLGDYVD RGYFSIECVL YLWVLKILYP STLFLLRGNH ECRHLTEYFT FKQECKIKYS

181 ERVYEACMEA FDSLPLAALL NQQFLCVHGG LSPEIHTLDD IRRLDRFKEP PAFGPMCDLL

241 WSDPSEDFGN EKSQEHFSHN TVRGCSYFYN YPAVCEFLQN NNLLSIIRAH EAQDAGYRMY

301 RKSQTTGFPS LITIFSAPNY LDVYNNKAAV LKYENNVMNI RQFNCSPHPY WLPNFMDVFT

361 WSLPFVGEKV TEMLVNVLSI CSDDELMTEG EDQFDGSAAA RKEIIRNKIR AIGKMARVFS

421 VLREESESVL TLKGLTPTGM LPSGVLAGGR QTLQSATVEA IEAEKAIRGF SPPHRICSFE

481 EAKGLDRINE RMPPRKDAVQ QDGFNSLNTA HATENHGTGN HTAQ*

Subunit # (isoform 2) of calcineurin A (512 AAs, NP_005596, GI:21361290, map

8p21.3, gene PPP3CC, Homo sapiens).

1 MSGRRFHLST TDRVIKAVPF PPTQRLTFKE VFENGKPKVD VLKNHLVKEG RLEEEVALKI

61 INDGAAILRQ EKTMIEVDAP ITVCGDIHGQ FFDLMKLFEV GGSPSNTRYL FLGDYVDRGY

121 FSIECVLYLW SLKINHPKTL FLLRGNHECR HLTDYFTFKQ ECRIKYSEQV YDACMETFDC

181 LPLAALLNQQ FLCVHGGMSP EITSLDDIRK LDRFTEPPAF GPVCDLLWSD PSEDYGNEKT

241 LEHYTHNTVR GCSYFYSYPA VCEFLQNNNL LSIIRAHEAQ DAGYRMYRKS QATGFPSLIT

301 IFSAPNYLDV YNNKAAVLKY ENNVMNIRQF NCSPHPYWLP NFMDVFTWSL PFVGEKVTEM

361 LVNVLNICSD DELISDDEAE GSTTVRKEII RNKIRAIGKM ARVFSILRQE SESVLTLKGL

421 TPTGTLPLGV LSGGKQTIET ATVEAVEARE AIRGFSLQHK IRSFEEARGL DRINERMPPR

481 KDSIHAGGPM KSVTSAHSHA AHRSDQGKKA HS*

1AUI 1M8F

1TC0

2P6B

Fig. Fs10

Figure Fs10. Comparative picture contains four X-ray structures 1AUI.pdb, 1M8F.pdb,

1TCO.pdb and 2P6B.pdb. The following entries are shown: 1AUI.pdb entitled ‘The human

calcineurin heterodimer’ [57]; resolution 2.10 Å; 2P6B.pdb entitled ‘Crystal structure of

human calcineurin in complex with PVIVIT peptide’ (calcineurin A, from 1 to 381 AAs),

calcineurin B (from 16 to 170 AAs) the sequence of the peptide is GPHPVIVITGPHEE-NH2,

resolution 2.30 Å [155]; 1MF8.pdb entitled ‘Crystal structure of human calcineurin

complexed with cyclosporin A and human cyclophilin’, calcineurin A (residues 20-392)

calcineurin B (residues 5-168) and human cyclophilin A (residues 1-164); resolution 3.10 Å

[Rs9]; 1TCO.pdb entitled ‘Ternary complex of calcineurin A fragment, calcineurin B, bovine

FKBP12 and the immunosuppressant drug FK506 (tacrolimus)’, calcinurin A (residues from

18 to 392 AAs), calcineurin B (residues from 1 to 169 AAs), and bFKBP12 [56].

Rs9. Jin L, Harrison SC (2002) Crystal structure of human calcineurin complexed with Cyclosporin A and human cyclophilin. Proc Natl Acad Sci USA 99:13522-1352

1

Figure Fs11. MSA of the human NF-ATs. NP_036472 ------------------------------------------------------------ NP_765978 ------------------------------------------------------------ NP_004546 ------------------------------------------------------------ NP_004545 ------------------------------------------------------------ NP_006590 MPSDFISLLSADLDLESPKSLYSRESVYDLLPKELQLPPSRETSVASMSQTSGGEAGSPP NP_619728 ------------------------------------------------------------ NP_036472 ------------------------------------MNAPERQPQPDGGDAPGHEPGGSP NP_765978 -------------------------------MPSTSFPVPSKFPLGPAAAVFGRGETLGP NP_004546 -------------------------------MTTANCGAHDELDFKLVFGEDGAPAPPPP NP_004545 -------------------------------MGAASC-EDEELEFKLVFGEEKEAPPLGA NP_006590 PAVVAADASSAPSSSSMGGACSSFTTSSSPTIYSTSVTDSKAMQVESCSSAVGVSNRGVS NP_619728 ----------------MGGACSSFTTSSSPTIYSTSVTDSKAMQVESCSSAVGVSNRGVS . . NP_036472 QDELDFSILFDYEYLNP----NEEEPNAHKVASPPSGPAYPDDVLDYGLKPYSPLASLSG NP_765978 APRAGGTMKSAEEEHYG----YASSNVSPALPLPTAHSTLPAPCHN--LQTSTPGIIPPA NP_004546 GSRPADLEPDDCASIYI----FNVDPPPSTLTTPLCLPHHGLPSHSSVLSPSFQLQSHKN NP_004545 GGLGEELDSEDAPPCCR----LALGEPPPYGAAPIGIPRPPPPRPGMHSPPPRPAPSPGT NP_006590 EKQLTSNTVQQHPSTPKRHTVLYISPPPEDLLDNSRMSCQDEGCGLESEQSCSMWMEDSP NP_619728 EKQLTSNTVQQHPSTPKRHTVLYISPPPEDLLDNSRMSCQDEGCGLESEQSCSMWMEDSP . . . NP_036472 EPPGRFGEPDRVGPQ-KFLSA--AKPAGASGL-SPRIEITPSHELIQAVGPLRMRDAGLL NP_765978 DHPSGYGAALDGGPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFH---DVE NP_004546 YEGTCEIPESKYSPLGGP------KPFECPSIQITSISPNCHQELDAHEDDLQINDPERE NP_004545 WESQPARSVRLGGPGGGAGGAGGGRVLECPSIRITSISPTPEPPAALEDNPDAWGDGSPR NP_006590 SNFSNMSTSSYNDNTEVPRKSRKRNPKQRPGVKRRDCEESNMDIFDADSAKAPHYVLSQL NP_619728 SNFSNMSTSSYNDNTEVPRKSRKRNPKQRPGVKRRDCEESNMDIFDADSAKAPHYVLSQL . . ..: . . NP_036472 VEQPP--------------LAGVAASPRFTLPVPGFEGYREPLCLSPASSGSSASFISDT NP_765978 VEDVL--------------PSSKRSPSTATLSLPSLEAYRDPSCLSPASSLSSRSCNSEA NP_004546 FLERPSRDHL-------YLPLEPSYRESSLSPSP-ASSISSRSWFSDASSCESLSHIYDD NP_004545 DYPPPEGFGG-------YREAGGQGGGAFFSPSPGSSSLSSWSFFSDASDEAALYAACDE NP_006590 TTDNKGNSKAGNGTLENQKGTGVKKSPMLCGQYPVKSEGKELKIVVQPETQHRARYLTEG NP_619728 TTDNKGNSKAGNGTLENQKGTGVKKSPMLCGQYPVKSEGKELKIVVQPETQHRARYLTEG * . . . .. : NP_036472 -----------------FSPYTSPCVSPNNGGPDDLCPQ-FQNIPAHYSPRTSPIMSPRT NP_765978 SSYESNY--SYPYASPQTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPSTSPRA NP_004546 VDSELN---EAAARFTLGSPLTSPGGSPGGCPGEETWHQQYGLGHSL-SPRQSPCHSPRS NP_004545 VESELN---EAASRFGLGSPLPSPRASPRPWTPEDPWS-LYG-----------PSPGGRG NP_006590 SRGSVKDRTQQGFPTVKLEGHNEPVVLQVFVGNDSGRVKPHGFYQACRVTGRNTTPCKEV NP_619728 SRGSVKDRTQQGFPTVKLEGHNEPVVLQVFVGNDSGRVKPHGFYQACRVTGRNTTPCKEV . .* :. . . NP_036472 SLAEDSCLGRHSPVPRPASRSSSPGAKRRHSCAEALVALPPGASPQRSRSP--------S NP_765978 SVTEESWLGARS------SRPASPCNKRKYS----LNGRQPPYSPHHSPTP--------S NP_004546 SVTDENWLSPRPASG-PSSRPTSPCGKRRHSS--AEVCYAGSLSPHHSPVP--------S NP_004545 PEDSWLLLSAPGPTP-ASPRPASPCGKRRYSS--SGT--PSSASPALSRRG--------S NP_006590 DIEGTTVIEVGLDPSNNMTLAVDCVGILKLRNADVEARIGIAGSKKKSTRARLVFRVNIM NP_619728 DIEGTTVIEVGLDPSNNMTLAVDCVGILKLRNADVEARIGIAGSKKKSTRARLVFRVNIM : . . . : * *

2

NP_036472 PQPSSHVAPQDHGSPAGYPPVAGSAVIMDALNSLATDS----PCGIPPKMWK-------- NP_765978 PHGSPRVSVTDD-SWLGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRK-------- NP_004546 PGHSPRGSVTEDTWLNASVHGGSGLGPAVFPFQYCVETD------IPLKTRK-------- NP_004545 LGEE--GSEPPPPPPLPLARDPGSPGPFDYVGAPPAES-------IPQKTRR-------- NP_006590 RKDGSTLTLQTPSSPILCTQPAGVPEILKKSLHSCSVKGEEEVFLIGKNFLKGTKVIFQE NP_619728 RKDGSTLTLQTPSSPILCTQPAGVPEILKKSLHSCSVKGEEEVFLIGKNFLKGTKVIFQE : . : : : NP_036472 -----TSPD-PSPVSAAPSKAGLPRHIYPA-VEFLGPCEQG---ERRNSAPESILLVPPT NP_765978 -----TTLEQPPSVALKVEPVGEDLGSPPP-PADFAPEDYSSFQHIRKGGFCDQYLAVPQ NP_004546 -----TSEDQAAILPGKLELCSDDQGSLSPARETSIDDGLGSQYPLKKDSCGDQFLSVPS NP_004545 -----TSSEQAVALPRSEEPASCN-GKLPLGAEESVAPPGGS----RKEVAGMDYLAVPS NP_006590 NVSDENSWKSEAEIDMELFHQNHLIVKVPPYHDQHITLPVSVGIYVVTNAGRSHDVQPFT NP_619728 NVSDENSWKSEAEIDMELFHQNHLIVKVPPYHDQHITLPVSVGIYVVTNAGRSHDVQPFT .: . : . . . . : NP_036472 WPKPLVPAIPICSIP-VTASLPPLEWPLSSQSGSYELR---------------------- NP_765978 HPYQWAKPKPLSPTSYMSPTLPALDWQLPSHSGPYELR---------------------- NP_004546 PFTWSKPKPG-HTPIFRTSSLPPLDWPLPAHFGQCELK---------------------- NP_004545 PLAWSKARIGGHSPIFRTSALPPLDWPLPSQYEQLELR---------------------- NP_006590 YTPDPAAAGALNVNVKKEISSPARPCSFEEAMKAMKTTGCNLDKVNIIPNALMTPLIPSS NP_619728 YTPDPAAAGALNVNVKKEISSPARPCSFEEAMKAMKTTGCNLDKVNIIPNALMTPLIPSS : *. : : NP_036472 ---------IEVQPKPHHRAHYETEGSRGAVKAPTGGHPVVQLHGYMENKPLGLQIFIGT NP_765978 ---------IEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGT NP_004546 ---------IEVQPKTHHRAHYETEGSRGAVKASTGGHPVVKLLGYNE-KPINLQMFIGT NP_004545 ---------IEVQPRAHHRAHYETEGSRGAVKAAPGGHPVVKLLGYSE-KPLTLQMFIGT NP_006590 MIKSEDVTPMEVTAEKRSSTIFKTTKSVGSTQQTLENISNIAGNGSFS-SPSSSHLPSEN NP_619728 MIKSEDVTPMEVTAEKRSSTIFKTTKSVGSTQQTLENISNIAGNGSFS-SPSSSHLPSEN :** .. : : ::* * *:.: . . . : * . .* :: . NP_036472 ADERILKPHAFY-------QVHRITG--------------------------KTVTTTSY NP_765978 ADDRLLRPHAFY-------QVHRITG--------------------------KTVSTTSH NP_004546 ADDRYLRPHAFY-------QVHRITG--------------------------KTVATASQ NP_004545 ADERNLRPHAFY-------QVHRITG--------------------------KMVATASY NP_006590 EKQQQIQPKAYNPETLTTIQTQDISQPGTFPAVSASSQLPNSDALLQQATQFQTRETQSR NP_619728 EKQQQIQPKAYNPETLTTIQTQDISQPGTFPAVSASSQLPNSDALLQQATQFQTRETQSR .:: ::*:*: *.: *: : * * NP_036472 EKIVGNTKVLEIPLEPKNN--------------------------------MRATIDCAG NP_765978 EAILSNTKVLEIPLLPENS--------------------------------MRAVIDCAG NP_004546 EIIIASTKVLEIPLLPENN--------------------------------MSASIDCAG NP_004545 EAVVSGTKVLEMTLLPENN--------------------------------MAANIDCAG NP_006590 EILQSDGTVVNLSQLTEASQQQQQSPLQEQAQTLQQQISSNIFPSPNSVSQLQNTIQQLQ NP_619728 EILQSDGTVVNLSQLTEASQQQQQSPLQEQAQTLQQQISSNIFPSPNSVSQLQNTIQQLQ * : .. .*:::. .: . : *: NP_036472 ILKLRNADIELRKGETDIGRKNTRVRLVFR---VHIPESSGRIVSLQTASNPIECSQRSA NP_765978 ILKLRNSDIELRKGETDIGRKNTRVRLVFR---VHVPQPSGRTLSLQVASNPIECSQRSA NP_004546 ILKLRNSDIELRKGETDIGRKNTRVRLVFR---VHIPQPSGKVLSLQIASIPVECSQRSA NP_004545 ILKLRNSDIELRKGETDIGRKNTRVRLVFR---VHVPQGGGKVVSVQAASVPIECSQRSA NP_006590 AGSFTGSTASGSSGSVDLVQQVLEAQQQLSSVLFSAPDGNENVQEQLSADIFQQVSQIQS NP_619728 AGSFTGSTASGSSGSVDLVQQVLEAQQQLSSVLFSAPDGNENVQEQLSADIFQQVSQIQS .: .: . .*..*: :: ..: : . *: . . . *. : ** .: NP_036472 HELPMVERQDTDSCLVYGGQQMILTGQNFTSESKVVFTEKTTDGQQI-----------WE NP_765978 QELPLVEKQSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHV-----------WE NP_004546 QELPHIEKYSINSCSVNGGHEMVVTGSNFLPESKIIFLEKGQDGRPQ-----------WE NP_004545 QELPQVEAYSPSACSVRGGEELVLTGSNFLPDSKVVFIERGPDGKLQ-----------WE NP_006590 GVSPGMFSSTEPTVHTRPDNLLPGRAESVHPQSENTLSNQQQQQQQQQQVMESSAAMVME NP_619728 GVSPGMFSSTEPTVHTRPDNLLPGRAESVHPQSENTLSNQQQQQQQQQQVMESSAAMVME * : : . .. : . .. :*: : :: : : *

3

NP_036472 MEATVDKDKSQPNMLFVEIPEYRN------KHIRTPVKVNFYVINGKRKRSQPQHFTYHP NP_765978 MEAKTDRDLCKPNSLVVEIPPFRN------QRITSPVHVSFYVCNGKRKRSQYQRFTYLP NP_004546 VEGKIIREKCQGAHIVLEVPPYHN------PAVTAAVQVHFYLCNGKRKKSQSQRFTYTP NP_004545 EEATVNRLQSNEVTLTLTVPEYSN------KRVSRPVQVYFYVSNGRRKRSPTQSFRFLP NP_006590 MQQSICQAAAQIQSELFPSTASANGNLQQSPVYQQTSHMMSALSTNEDMQMQCELFSSPP NP_619728 MQQSICQAAAQIQSELFPSTASANGNLQQSPVYQQTSHMMSALSTNEDMQMQCELFSSPP : . : .: . . * . :: : ... : : * * NP_036472 VPAIKT------------------------EPTDEYDPTLICSPTHGGLGSQPYYPQH-- NP_765978 A--------------------------------NGNAIFLTVSREHERVG---------- NP_004546 VLMKQEHREEIDLSSVPSLPVPHPAQTQRPSSDSGCSHDSVLSGQRSLICSIPQTYAS-- NP_004545 VICKEEPLPDSSLRGFP------------SASATPFGTDMDFSPPRPPYPSYPHEDP--- NP_006590 AVSGNETSTTTTQQVATPGTTMFQTSSSGDGEETGTQAKQIMGGACSSFTTSSSP----- NP_619728 AVSGNETSTTTTQQVATPGTTMFQTSSSGDGEETGTQAKQIQNSVFQTMVQMQHSGDNQP . . NP_036472 ---------PMVAESPSCLVATMAPCQQFRTGLSSPDARYQQQNPAAVLYQRSKSLSPSL NP_765978 -----------------CFF---------------------------------------- NP_004546 ---------MVTSSHLPQLQCRDESVSKEQHMIPSPIVHQPFQVTPTPPVGSSYQPMQTN NP_004545 -------------------ACETPYLS-EGFGYGMPPLYP--QTGPPP----SYRPGLR- NP_006590 --TIYSTSVTDSKAMQVESCSSAVGVSNRGVSEKQLTSNTVQQHPSTPKRHTVLYISPPP NP_619728 QVNLFSSTKSMMSVQNSGTQQQGNGLFQQGNEMMSLQSGNFLQQSS----HSQAQLFHPQ NP_036472 LGYQQPALMAAPLSLADAHR----------------------SVLVHAGSQGQSSALLHP NP_765978 ------------------------------------------------------------ NP_004546 VVYNGPTCLPINAASSQEFDSVLFQQDATLSGLVNLGCQPLSSIPFHSSNSGSTGHLLAH NP_004545 ---------------------------------------------MFPETRGTTG---CA NP_006590 EDLLDNSRMSCQDEGCGLESEQSCSMWMEDSPSNFSNMSTSSYNDNTEVPRKSRKRNPKQ NP_619728 NPIADAQNLSQETQGSLFHS-PNPIVHSQTSTTSSEQMQPPMFHSQSTIAVLQGSSVPQD NP_036472 SPTNQQASP--------------VIHYSPTNQQLRCGSHQEFQHIMYCENFAP-GTTRPG NP_765978 ------------------------------------------------------------ NP_004546 TPHSVHTLPHLQSMGYHCSNTGQRSLSSPVADQITGQPSSQLQPITYGPSHSGSATTASP NP_004545 QPPAVSFLP--------------RPFPS---DPYGGRGSS----FSLGLPFSPPAPFRPP NP_006590 RPGVKRRDCEESNMDIFDADSAKAPHYVLSQLTTDNKGNSKAG-NGTLENQKGTGVKKSP NP_619728 QQSTNIFLSQSPMNNLQTNTVAQEAFFAAPNSISPLQSTSNSEQQAAFQQQAPISHIQTP NP_036472 PPPVSQGQRLSPGSYPTVIQQQNATSQRAAKNGPPVSDQKEVLPAGVTIKQEQNLDQTYL NP_765978 ------------------------------------------------------------ NP_004546 AASHPLASSPLSGPPSPQLQPMPYQSPSSGTASSPSPATRMHSGQHSTQAQSTGQGGLSA NP_004545 PLP---ASPPLEGPFPSQSDVHPLPAEGYNKVGPG------YGPGEGAPEQEKSRGGYSS NP_006590 MLCGQYPVKSEGKELKIVVQPETQHRARYLTEGSRGSVKDRTQQGFPTVKLEGHNEPVVL NP_619728 MLSQEQAQPPQQG----LFQPQVALGSLPPNPMPQSQQGTMFQSQHSIVAMQSNSPSQEQ NP_036472 ------------------DDELIDTHLSWIQNIL-------------------------- NP_765978 ------------------------------------------------------------ NP_004546 PSSLICHSLCDPMGAASCEDEELEFKLVFGEEKEAPPLGAGGLGEELDSEDAPPCCRLAL NP_004545 ----------------GFRDSVPIQGITLEEVSEI-------IGRDLSGFPAPP------ NP_006590 QVFVGNDSGRVKPHGFYQACRVTGRNTTPCKEVDIEGTTVIEVGLDPSNNMTLAVDCVGI NP_619728 QQQQQQQQQQQQQQQQSILFSNQNTMATMASPKQPPPNMIFNPNQNPMANQEQQNQSIFH NP_036472 ------------------------------------------------------------ NP_765978 ------------------------------------------------------------ NP_004546 GEPPPYGAAPIGIPRPPPPR---------------------------------------- NP_004545 GEEPPA------------------------------------------------------ NP_006590 LKLRNADVEARIGIAGSKKKSTRARLVFRVNIMRKDGSTLTLQTPSSPILCTQPAGVPEI NP_619728 QQSNMAPMNQEQQPMQFQSQSTVSSLQNPGPTQSESSQTPLFHSSPQIQLVQGSPSSQEQ

4

NP_036472 ------------------------------------------------------------ NP_765978 ------------------------------------------------------------ NP_004546 ------------------------------------------------------------ NP_004545 ------------------------------------------------------------ NP_006590 LKKSLHSCSVKGEEEVFLIGKNFLKGTKVIFQENVSDENSWKSEAEIDMELFHQNHLIVK NP_619728 QVTLFLSPASMSALQTSINQQDMQQSPLYSPQNNMPGIQGATSSPQPQATLFHN-TAGGT NP_036472 ------------------------------------------------------------ NP_765978 ------------------------------------------------------------ NP_004546 ------------------------------------------------------------ NP_004545 ------------------------------------------------------------ NP_006590 VPPYHDQHITLPVSVGIYVVTNAGRSHDVQPFTYTPDPAAAGALNVNVKKEISSPARPCS NP_619728 MNQLQNSPGSSQQTSGMFLFG--IQNNCSQLLTSGPATLPDQLMAISQPGQPQNEGQPPV NP_036472 -------------------------------------------- NP_765978 -------------------------------------------- NP_004546 -------------------------------------------- NP_004545 -------------------------------------------- NP_006590 FEEAMKAMKTTGCNLDKVNIIPNALMTPLIPSSMIKSEDV---- NP_619728 TTLLSQQMPENSPLASSINTNQNIEKIDLLVSLQNQGNNLTGSF

Fig. Fs12 Alignment of TOR1/TOR2 from Saccharomyces cerevisiae Sequence indexes are for ScTOR1 (P35169; upper sequence) 10 19 29 39 49 59 P35169 MEPHEEQIWK S-KLLKAANN DMDMDRNVPL APNLNVNMNM KMNASRNGDE FGLTSSRFDG P32600 MNKYINKYTT PPNLLSLRQR AEGKHRTRKK LTHKSHSHDD EMSTTSNTDS NHNGPNDSGR *: : :: . . :**. :. . .*. .: . . : :*.:: * *. .. . 66 72 78 88 98 108 P35169 VVIGSN---G DVNFKP---- ----ILEKIF RELTSDYKEE RKLASISLFD LLVSLEHELS P32600 VITGSAGHIG KISFVDSELD TTFSTLNLIF DKLKSDVPQE RASGANELST TLTSLAREVS *: ** * .:.* *: ** :*.** :* * .: .* *.** :*:* 118 128 138 148 158 168 P35169 IEEFQAVSND INNKILELVH TKKTSTRVGA VLSIDTLISF YAYTERLPNE TSRLAGYLRG P32600 AEQFQRFSNS LNNKIFELIH GFTSSEKIGG ILAVDTLISF YLSTEELPNQ TSRLANYLRV *:** .**. :****:**:* .:* ::*. :*::****** * **.***: *****.*** 178 188 198 208 218 228 P35169 LIPSNDVEVM RLAAKTLGKL AVPGGTYTSD FVEFEIKSCL EWLTASTEKN SFSSSKPDHA P32600 LIPSSDIEVM RLAANTLGRL TVPGGTLTSD FVEFEVRTCI DWLTLTADNN S-SSSKLEYR ****.*:*** ****:***:* :***** *** *****:::*: :*** ::::* * **** :: 238 248 258 268 278 288 P35169 KHAALLIITA LAENCPYLLY QYLNSILDNI WRALRDPHLV IRIDASITLA KCLSTLRNRD P32600 RHAALLIIKA LADNSPYLLY PYVNSILDNI WVPLRDAKLI IRLDAAVALG KCLTIIQDRD :*******.* **:*.***** *:******* * .***.:*: **:**:::*. ***: :::** 298 308 318 328 338 348 P35169 PQLTSQWVQR LATSCEYGFQ VNTLECIHAS LLVYKEILFL KDPFLNQVFD QMCLNCIAYE P32600 PALGKQWFQR LFQGCTHGLS LNTNDSVHAT LLVFRELLSL KAPYLRDKYD DIYKSTMKYK * * .**.** * .* :*:. :** :.:**: ***::*:* * * *:*.: :* :: . : *:

358 368 378 388 398 408

P35169 NHKAKMIREK IYQIVPLLAS FNPQLFAGKY LHQIMDNYLE ILTNAPANKI PHLKDDKPQI

P32600 EYKFDVIRRE VYAILPLLAA FDPAIFTKKY LDRIMVHYLR YLKNIDMNAA NNS--DKPFI

::* .:**.: :* *:****: *:* :*: ** *.:** :**. *.* * : *** *

418 428 438 448 458 468

P35169 LISIGDIAYE VGPDIAPYVK QILDYIEHDL QTKFKFRKKF ENEIFYCIGR LAVPLGPVLG

P32600 LVSIGDIAFE VGSSISPYMT LILDNIREGL RTKFKVRKQF EKDLFYCIGK LACALGPAFA

*:******:* **..*:**:. *** *...* :****.**:* *:::*****: ** .***.:.

478 488 498 508 518 528

P35169 KLLNRNILDL MFKCPLSDYM QETFQILTER IPSLGPKIND ELLNLVCSTL SGTPFIQPGS

P32600 KHLNKDLLNL MLNCPMSDHM QETLMILNEK IPSLESTVNS RILNLLSISL SGEKFIQSNQ

* **:::*:* *::**:**:* ***: **.*: **** ..:*. .:***:. :* ** ***...

538 548 558 568 578 588

P35169 PMEIPSFSRE RAREWRNKNI LQKTGESNDD NNDIKIIIQA FRMLKNIKSR FSLVEFVRIV

P32600 YDFNNQFSIE KARKSRNQSF MKKTGESNDD ITDAQILIQC FKMLQLIHHQ YSLTEFVRLI

.** * :**: **:.: ::******** .* :*:**. *:**: *: : :**.****::

598 608 618 628 638 648

P35169 ALSYIEHTDP RVRKLAALTS CEIYVKDNIC KQTSLHSLNT VSEVLSKLLA ITIADPLQDI

P32600 TISYIEHEDS SVRKLAALTS CDLFIKDDIC KQTSVHALHS VSEVLSKLLM IAITDPVAEI

::***** *. ********* *::::**:** ****:*:*:: ********* *:*:**: :*

658 668 678 688 698 708

P35169 RLEVLKNLNP CFDPQLAQPD NLRLLFTALH DESFNIQSVA MELVGRLSSV NPAYVIPSIR

P32600 RLEILQHLGS NFDPQLAQPD NLRLLFMALN DEIFGIQLEA IKIIGRLSSV NPAYVVPSLR

***:*::*.. ********* ****** **: ** *.** * ::::****** *****:**:*

718 728 738 748 758 768

P35169 KILLELLTKL KFSTSSREKE ETASLLCTLI RSSKDVAKPY IEPLLNVLLP KFQDTSSTVA

P32600 KTLLELLTQL KFSNMPKKKE ESATLLCTLI NSSDEVAKPY IDPILDVILP KCQDASSAVA

* ******:* ***. .::** *:*:****** .**.:***** *:*:*:*:** * **:**:**

778 788 798 808 818 828

P35169 STALRTIGEL SVVGGEDMKI YLKDLFPLII KTFQDQSNSF KREAALKALG QLAASSGYVI

P32600 STALKVLGEL SVVGGKEMTR YLKELMPLII NTFQDQSNSF KRDAALTTLG QLAASSGYVV

****:.:*** *****::*. ***:*:**** :********* **:***.:** *********:

838 848 858 868 878 888

P35169 DPLLDYPELL GILVNILKTE NSQNIRRQTV TLIGILGAID PYRQKEREVT STTDISTEQN

P32600 GPLLDYPELL GILINILKTE NNPHIRRGTV RLIGILGALD PYKHREIEVT SNSKSSVEQN

.********* ***:****** *. :*** ** *******:* **:::* *** *.:. *.***

898 908 918 928 938 948

P35169 APPIDIALLM QGMSPSNDEY YTTVVIHCLL KILKDPSLSS YHTAVIQAIM HIFQTLGLKC

P32600 APSIDIALLM QGVSPSNDEY YPTVVIHNLM KILNDPSLSI HHTAAIQAIM HIFQNLGLRC

**.******* **:******* *.***** *: ***:***** :***.***** ****.***:*

958 968 978 988 998 1008

P35169 VSFLDQIIPT ILDVMRTCSQ SLLEFYFQQL CSLIIIVRQH IRPHVDSIFQ AIKDFSSVAK

P32600 VSFLDQIIPG IILVMRSCPP SQLDFYFQQL GSLISIVKQH IRPHVEKIYG VIREFFPIIK

********* *: ***:*. * *:****** *** **:** *****:.*: .*::* .: *

1018 1028 1038 1048 1058 1068

P35169 LQITLVSVIE AISKALEGEF KRLVPLTLTL FLVILENDKS SDKVLSRRVL RLLESFGPNL

P32600 LQITIISVIE SISKALEGEF KRFVPETLTF FLDILENDQS NKRIVPIRIL KSLVTFGPNL

****::**** :********* **:** ***: ** *****:* ..:::. *:* : * :*****

1078 1088 1098 1108 1118 1128

P35169 EGYSHLITPK IVQMAEFTSG NLQRSAIITI GKLAKDVDLF EMSSRIVHSL LRVLSSTTSD

P32600 EDYSHLIMPI VVRMTEYSAG SLKKISIITL GRLAKNINLS EMSSRIVQAL VRILNNGD-R

*.***** * :*:*:*:::* .*:: :***: *:***:::* *******::* :*:*..

1138 1148 1158 1168 1178 1188

P35169 ELSKVIMNTL SLLLIQMGTS FAIFIPVINE VLMKKHIQHT IYDDLTNRIL NNDVLPTKIL

P32600 ELTKATMNTL SLLLLQLGTD FVVFVPVINK ALLRNRIQHS VYDQLVNKLL NNECLPTNII

**:*. **** ****:*:**. *.:*:****: .*::::***: :**:*.*::* **: ***:*:

1988 1108 1218 1228 1238 1248

P35169 EANTTDYKPA EQMEAADAGV AKLPINQSVL KSAWNSSQQR TKEDWQEWSK RLSIQLLKES

P32600 FDKENE-VPE RKNYEDEMQV TKLPVNQNIL KNAWYCSQQK TKEDWQEWIR RLSIQLLKES

: .: * .: : * :***:**.:* *.** .***: ******** : **********

1258 1268 1278 1288 1298 1308

P35169 PSHALRACSN LASMYYPLAK ELFNTAFACV WTELYSQYQE DLIGSLCIAL SSPLNPPEIH

P32600 PSACLRSCSS LVSVYYPLAR ELFNASFSSC WVELQTSYQE DLIQALCKAL SSSENPPEIY

** .**:**. *.*:*****: ****::*:. *.** :.*** *** :** ** **. *****:

1318 1328 1338 1348 1358 1368

P35169 QTLLNLVEFM EHDDKALPIP TQSLGEYAER CHAYAKALHY KEIKFIKEPE NSTIESLISI

P32600 QMLLNLVEFM EHDDKPLPIP IHTLGKYAQK CHAFAKALHY KEVEFLEEPK NSTIEALISI

* ******** *****.**** ::**:**:: ***:****** **::*::**: *****:****

1378 1388 1398 1408 1418 1428

P35169 NNQLNQTDAA IGILKHAQQH HSLQLKETWF EKLERWEDAL HAYNEREKAG DTSVSVTLGK

P32600 NNQLHQTDSA IGILKHAQQH NELQLKETWY EKLQRWEDAL AAYNEKEAAG EDSVEVMMGK

****:***:* ********** :.*******: ***:****** ****:* ** : **.* :**

1438 1448 1458 1468 1478 1488

P35169 MRSLHALGEW EQLSQLAARK WKVSKLQTKK LIAPLAAGAA WGLGEWDMLE QYISVMKPKS

P32600 LRSLYALGEW EELSKLASEK WGTAKPEVKK AMAPLAAGAA WGLEQWDEIA QYTSVMKSQS

:***:***** *:**:**:.* * .:* :.** :******** *** :** : ** ****.:*

1498 1508 1518 1528 1538 1548

P35169 PDKEFFDAIL YLHKNDYDNA SKHILNARDL LVTEISALIN ESYNRAYSVI VRTQIITEFE

P32600 PDKEFYDAIL CLHRNNFKKA EVHIFNARDL LVTELSALVN ESYNRAYNVV VRAQIIAELE

*****:**** **:*::.:* . **:***** ****:***:* *******.*: **:***:*:*

1558 1568 1578 1588 1598 1608

P35169 EIIKYKQLPP NSEKKLHYQN LWTKRLLGCQ KNVDLWQRVL RVRSLVIKPK QDLQIWIKFA

P32600 EIIKYKKLPQ NSDKRLTMRE TWNTRLLGCQ KNIDVWQRIL RVRSLVIKPK EDAQVRIKFA

******:** **:*:* :: *..****** **:*:***:* ********** :* *: ****

1618 1628 1638 1648 1658 1668

P35169 NLCRKSGRMR LANKALNMLL EGGNDPSLPN TFKAPPPVVY AQLKYIWATG AYKEALNHLI

P32600 NLCRKSGRMA LAKKVLNTLL EETDDPDHPN TAKASPPVVY AQLKYLWATG LQDEALKQLI

********* **:*.** ** * :**. ** * **.***** *****:**** .***::**

1678 1688 1698 1708 1718 1728

P35169 GFTSRLAHDL GLDPNNMIAQ SVKLSSASTA PYVEEYTKLL ARCFLKQGEW RIATQPNWRN

P32600 NFTSRMAHDL GLDPNNMIAQ SVPQQSKRVP RHVEDYTKLL ARCFLKQGEW RVCLQPKWRL

.****:**** ********** ** .* .. :**:***** ********** *:. **:**

1738 1748 1758 1768 1778 1788

P35169 TNPDAILGSY LLATHFDKNW YKAWHNWALA NFEVISMVQE ETKLNGGKND DDDDTAVNND

P32600 SNPDSILGSY LLATHFDNTW YKAWHNWALA NFEVISMLTS VSKKKQEGSD ASSVTDIN--

:***:***** *******:.* ********** *******: . :* : .* .. * :*

1798 1808 1818 1828 1838 1848

P35169 NVRIDGSILG SGSLTINGNR YPLELIQRHV VPAIKGFFHS ISLLETSCLQ DTLRLLTLLF

P32600 --EFDNGMIG VNTFDAKEVH YSSNLIHRHV IPAIKGFFHS ISLSESSSLQ DALRLLTLWF

.:*..::* .:: : : *. :**:*** :********* *** *:*.** *:****** *

1858 1868 1878 1888 1898 1908

P35169 NFGGIKEVSQ AMYEGFNLMK IENWLEVLPQ LISRIHQPDP TVSNSLLSLL SDLGKAHPQA

P32600 TFGGIPEATQ AMHEGFNLIQ IGTWLEVLPQ LISRIHQPNQ IVSRSLLSLL SDLGKAHPQA

.**** *.:* **:*****:: * .******* ********: **.****** **********

1918 1928 1938 1948 1958 1968

P35169 LVYPLTVAIK SESVSRQKAA LSIIEKIRIH SPVLVNQAEL VSHELIRVAV LWHELWYEGL

P32600 LVYPLMVAIK SESLSRQKAA LSIIEKMRIH SPVLVDQAEL VSHELIRMAV LWHEQWYEGL

***** **** ***:****** ******:*** *****:**** *******:** **** *****

1978 1988 1998 2008 2018 2028

P35169 EDASRQFFVE HNIEKMFSTL EPLHKHLGNE PQTLSEVSFQ KSFGRDLNDA YEWLNNYKKS

P32600 DDASRQFFGE HNTEKMFAAL EPLYEMLKRG PETLREISFQ NSFGRDLNDA YEWLMNYKKS

:******* * ** ****::* ***:: * . *:** *:*** :********* **** *****

2038 2048 2058 2068 2078 2087

P35169 KDINNLNQAW DIYYNVFRKI TRQIPQLQTL DLQHVSPQLL ATHDLELAVP GTYFPG-KPT

P32600 KDVSNLNQAW DIYYNVFRKI GKQLPQLQTL ELQHVSPKLL SAHDLELAVP GTRASGGKPI

**:.****** ********** :*:****** :******:** ::******** ** .* **

2097 2107 2117 2127 2137 2147

P35169 IRIAKFEPLF SVISSKQRPR KFSIKGSDGK DYKYVLKGHE DIRQDSLVMQ LFGLVNTLLK

P32600 VKISKFEPVF SVISSKQRPR KFCIKGSDGK DYKYVLKGHE DIRQDSLVMQ LFGLVNTLLQ

::*:****:* ********** **.******* ********** ********** *********:

2157 2167 2177 2187 2197 2207

P35169 NDSECFKRHL DIQQYPAIPL SPKSGLLGWV PNSDTFHVLI REHRDAKKIP LNIEHWVMLQ

P32600 NDAECFRRHL DIQQYPAIPL SPKSGLLGWV PNSDTFHVLI REHREAKKIP LNIEHWVMLQ

**:***:*** ********** ********** ********** ****:***** **********

2217 2227 2237 2247 2257 2267

P35169 MAPDYENLTL LQKIEVFTYA LDNTKGQDLY KILWLKSRSS ETWLERRTTY TRSLAVMSMT

P32600 MAPDYDNLTL LQKVEVFTYA LNNTEGQDLY KVLWLKSRSS ETWLERRTTY TRSLAVMSMT

*****:**** ***:****** *:**:***** *:******** ********** **********

2277 2287 2297 2307 2317 2327

P35169 GYILGLGDRH PSNLMLDRIT GKVIHIDFGD CFEAAILREK YPEKVPFRLT RMLTYAMEVS

P32600 GYILGLGDRH PSNLMLDRIT GKVIHIDFGD CFEAAILREK FPEKVPFRLT RMLTYAMEVS

********** ********** ********** ********** :********* **********

2337 2347 2357 2367 2377 2387

P35169 GIEGSFRITC ENVMRVLRDN KESLMAILEA FALDPLIHWG FDLPPQKLTE QTGIPLPLIN

P32600 GIEGSFRITC ENVMKVLRDN KGSLMAILEA FAFDPLINWG FDLPTKKIEE ETGIQLPVMN

********** ****:***** * ******** **:****:** ****.:*: * :*** **::*

2397 2407 2417 2427 2437 2447

P35169 PSELLRKGAI TVEEAANMEA EQQNETKNAR AMLVLRRITD KLTGNDIKRF NELDVPEQVD

P32600 ANELLSNGAI TEEEVQRVEN EHKNAIRNAR AMLVLKRITD KLTGNDIRRF NDLDVPEQVD

..*** :*** * **. .:* *::* :*** *****:**** *******:** *:********

2457 2467 P35169 KLIQQATSIE RLCQHYIGWC PFW-2470 P32600 KLIQQATSVE NLCQHYIGWC PFW-2474 ********:* .********* ***

Domain HEAT from 1 to 11 are in red ink (eg. HEAT11 1109-1147) Domain FAT is in brown ink (1331-1919 AAs) Domain Rapamycin-binding in pink ink (1755-2157 AAs) Domain PI3K/PI4K in green ink (2125-2437 AAs) Domain FATC in blue ink (2440-2460 AAs)

Figure Fs13 Alignment of ScTOR1 (P35169) with human TOR1 (NP_004949; ID = 39%) P35169 MEPHEEQIWKSKLLKAANNDMDMDRNVPLAPNLNVNMNMKMNASRNGDEFGLTSSRFDGV 60 NP_004949 --------------------------------------MLGTGPAAATTAATTSSNVS-- * ... . . ***... P35169 VIGSNGDVNFKPILEKIFRELTSDYKEERKLASISLFDLLVSLEHELSIEEFQAVSNDIN 120 NP_004949 ------------VLQQFASGLKSRNEETRAKAAKELQHYVTMELREMSQEESTRFYDQLN :*::: *.* :* * *: .* . :. :*:* ** . :::* P35169 NKILELVHTKKTSTRVGAVLSIDTLISFYAYTERLPNETSRLAGYLRGLIPSNDVEVMRL 180 NP_004949 HHIFELVSSSDANERKGGILAIASLIGVEGGNATR---IGRFANYLRNLLPSNDPVVMEM ::*:*** :..:. * *.:*:* :**.. . . .*:*.***.*:**** **.: P35169 AAKTLGKLAVPGGTYTSDFVEFEIKSCLEWLTASTEKNSFSSSKPDHAKHAALLIITALA 240 NP_004949 ASKAIGRLAMAGDTFTAEYVEFEVKRALEWLGADRNEG---------RRHAAVLVLRELA *:*::*:**:.*.*:*:::****:* .**** *. ::. :***:*:: ** P35169 ENCPYLLYQYLNSILDNIWRALRDPHLVIRIDASITLAKCLSTLRNRDPQ--LTSQWVQR 298 NP_004949 ISVPTFFFQQVQPFFDNIFVAVWDPKQAIREGAVAALRACLILTTQREPKEMQKPQWYRH . * :::* ::.::***: *: **: .** .* :* ** :*:*: ..** :: P35169 LATSCEYGF--------QVNTLECIHASLLVYKEILFLKDP------------------- 331 NP_004949 TFEEAEKGFDETLAKEKGMNRDDRIHGALLILNELVRISSMEGERLREEMEEITQQQLVH ..* ** :* : **.:**: :*:: :..

P35169 ------------------------------------------------------------ NP_004949 DKYCKDLMGFGTKPRHITPFTSFQAVQPQQSNALVGLLGYSSHQGLMGFGTSPSPAKSTL P35169 --------FLNQVFDQMCLNCIAYENHKAKMIREKIYQIVPLLASFNPQLFAG-KYLHQI 382 NP_004949 VESRCCRDLMEEKFDQVCQWVLKCRNSKNSLIQMTILNLLPRLAAFRPSAFTDTQYLQDT :::: ***:* : .* * .:*: .* :::* **:*.*. *:. :**:: P35169 MDNYLEILTNAPANKIPHLKDDKPQILISIGDIAYEVGPDIAPYVKQILDYIEHDLQTK- 441 NP_004949 MNHVLSCVKKE---------KERTAAFQALGLLSVAVRSEFKVYLPRVLDIIRAALPPKD *:: *. :.: .::. : ::* :: * .:: *: ::** *. * .* P35169 FKFRKK----FENEIFYCIGRLAVPLGPVLGKLLNRNILDLMFKCPLSDYMQETFQILTE 497 NP_004949 FAHKRQKAMQVDATVFTCISMLARAMGPGIQQDIK-ELLEPMLAVGLSPALTAVLYDLSR * .::: .: :* **. ** .:** : : :: ::*: *: ** : .: *:. P35169 RIPSLGPKINDELLNLVCSTLSGTPFIQPGSPMEIPSFSRERAREWRNKNILQKTGESND 557 NP_004949 QIPQLKKDIQDGLLKMLSLVLMHKPLRHPG-------MPKGLAHQLASPGLTTLPEAS-- :**.* .*:* **:::. .* .*: :** :.: *:: . .: . * P35169 DNNDIKIIIQAFRMLKNIKSR-FSLVEFVRIVALSYIEHTDPRVRKLAALTSC-----EI 611 NP_004949 ---DVGSITLALRTLGSFEFEGHSLTQFVRHCADHFLNSEHKEIRMEAARTCSRLLTPSI *: * *:* * .:: . .**.:*** * ::: . .:* ** *.. .* P35169 YVKDNICKQTSLHSLNTVSEVLSKLLAITIADPLQDIRLEVLKNLNPCFDPQLAQPDNLR 671 NP_004949 HLISGHAHVVSQTAVQVVADVLSKLLVVGITDPDPDIRYCVLASLDERFDAHLAQAENLQ :: .. .: .* :::.*::******.: *:** *** ** .*: **.:***.:**:

P35169 LLFTALHDESFNIQSVAMELVGRLSSVNPAYVIPSIRKILLELLTKLKFSTSSREKEETA 731 NP_004949 ALFVALNDQVFEIRELAICTVGRLSSMNPAFVMPFLRKMLIQILTELEHSGIGRIKEQSA **.**:*: *:*:.:*: ******:***:*:* :**:*:::**:*:.* .* **::* P35169 SLLCTLIRSSKDVAKPYIEPLLNVLLPKFQDTSS----TVASTALRTIGELSVVGGEDMK 787 NP_004949 RMLGHLVSNAPRLIRPYMEPILKALILKLKDPDPDPNPGVINNVLATIGELAQVSGLEMR :* *: .: : :**:**:*:.*: *::*... * ...* *****: *.* :*: P35169 IYLKDLFPLIIKTFQDQSNSFKREAALKALGQLAASSGYVIDPLLDYPELLGILVNILKT 847 NP_004949 KWVDELFIIIMDMLQDSSLLAKRQVALWTLGQLVASTGYVVEPYRKYPTLLEVLLNFLKT ::.:** :*:. :**.* **:.** :****.**:***::* .** ** :*:*:*** P35169 ENSQNIRRQTVTLIGILGAIDPYRQKE----------REVTSTTDISTEQNAPPIDIALL 896 NP_004949 EQNQGTRREAIRVLGLLGALDPYKHKVNIGMIDQSRDASAVSLSESKSSQDSSDYSTSEM *:.*. **::: ::*:***:***::* ...* :: .:.*::. . : : P35169 MQGMS-PSNDEYYTTVVIHCLLKILKDPSLSSYHTAVIQAIMHIFQTLGLKCVSFLDQII 955 NP_004949 LVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFIFKSLGLKCVQFLPQVM : .*. . **:*.:* : .*::*::* *** :** *:*** .**::******.** *:: P35169 PTILDVMRTCSQSLLEFYFQQLCSLIIIVRQHIRPHVDSIFQAIKDFSSVAK-LQITLVS 1014 NP_004949 PTFLNVIRVCDGAIREFLFQQLGMLVSFVKSHIRPYMDEIVTLMREFWVMNTSIQSTIIL **:*:*:*.*. :: ** **** *: :*:.****::*.*. :::* : . :* *:: P35169 VIEAISKALEGEFKRLVPLTLTLFLVILENDKSSDKVLSRRVLRLLESFGPNLEGYSHLI 1074 NP_004949 LIEQIVVALGGEFKLYLPQLIPHMLRVFMHDNSPGRIVSIKLLAAIQLFGANLDDYLHLL :** * ** **** :* :. :* :: :*:*..:::* ::* :: **.**:.* **:

P35169 TPKIVQMAEFTSGNLQR--SAIITIGKLAKDVDLFEMSSRIVHSLLRVLSSTTSDELSKV 1132 NP_004949 LPPIVKLFDAPEAPLPSRKAALETVDRLTESLDFTDYASRIIHPIVRTLD--QSPELRST * **:: : ... * :*: *:.:*::.:*: : :***:*.::*.*. * ** .. P35169 IMNTLSLLLIQMGTSFAIFIPVINEVLMKKHIQHTIYDDLTNRILNNDVLPTKILEANTT 1192 NP_004949 AMDTLSSLVFQLGKKYQIFIPMVNKVLVRHRINHQRYDVLICRIVKGYTLADEEEDPLIY *:*** *::*:*..: ****::*:**::::*:* ** * **::. .*. : :. P35169 DYKP--AEQMEAADAG------VAKLPINQSVLKSAWNSSQQRTKEDWQEWSKRLSIQLL 1244 NP_004949 QHRMLRSGQGDALASGPVETGPMKKLHVSTINLQKAWGAARRVSKDDWLEWLRRLSLELL ::: : * :* :* : ** :. *:.**.:::: :*:** ** :***::** P35169 KESPSHALRACSNLASMYYPLAKELFNTAFACVWTELYSQYQEDLIGSLCIALSSPLNPP 1304 NP_004949 KDSSSPSLRSCWALAQAYNPMARDLFNAAFVSCWSELNEDQQDELIRSIELALTS-QDIA *:*.* :**:* **. * *:*::***:**.. *:** .: *::** *: :**:* : . P35169 EIHQTLLNLVEFMEHDDK-ALPIPTQS----LGEYAERCHAYAKALHYKEIKFIKEPENS 1359 NP_004949 EVTQTLLNLAEFMEHSDKGPLPLRDDNGIVLLGERAAKCRAYAKALHYKELEFQKGPTPA *: ******.*****.** .**: :. *** * :*:**********::* * * : P35169 TIESLISINNQLNQTDAAIGILKHAQQHH-SLQLKETWFEKLERWEDALHAYNEREKAGD 1418 NP_004949 ILESLISINNKLQQPEAAAGVLEYAMKHFGELEIQATWYEKLHEWEDALVAYDKKMDTNK :********:*:*.:** *:*::* :*. .*::: **:***..***** **::: .:.. P35169 TSVSVTLGKMRSLHALGEWEQLSQLAARKWKVSKLQTKKLIAPLAAGAAWGLGEWDMLEQ 1478 NP_004949 DDPELMLGRMRCLEALGEWGQLHQQCCEKWTLVNDETQAKMARMAAAAAWGLGQWDSMEE . .: **:**.*.***** ** * ...**.: : :*: :* :**.******:** :*:

P35169 YISVMKPKSPDKEFFDAILYLHKNDYDNASKHILNARDLLVTEISALINESYNRAYSVIV 1538 NP_004949 YTCMIPRDTHDGAFYRAVLALHQDLFSLAQQCIDKARDLLDAELTAMAGESYSRAYGAMV * .:: .: * *: *:* **:: :. *.: * :***** :*::*: .***.***..:* P35169 RTQIITEFEEIIKYKQLPPNSEKKLHYQNLWTKRLLGCQKNVDLWQRVLRVRSLVIKPKQ 1598 NP_004949 SCHMLSELEEVIQYKLVP---ERREIIRQIWWERLQGCQRIVEDWQKILMVRSLVVSPHE ::::*:**:*:** :* *:: :::* :** ***: *: **::* *****:.*:: P35169 DLQIWIKFANLCRKSGRMRLANKALNMLLEGGNDPSLPNTFKAP-PPVVYAQLKYIWATG 1657 NP_004949 DMRTWLKYASLCGKSGRLALAHKTLVLLLGVDPSRQLDHPLPTVHPQVTYAYMKNMWKSA *:: *:*:*.** ****: **:*:* :** . . .* :.: : * *.** :* :* :. P35169 AYKEALNHLIGFTSRLAHDLGLDPNNMIAQSVKLSSASTAPYVEEYTKLLARCFLKQGEW 1717 NP_004949 RKIDAFQHMQHFVQ-----------TMQQQAQHAIATEDQQHKQELHKLMARCFLKLGEW :*::*: *.. .* *: : ::. : :* **:****** *** P35169 RIATQPNWRNTNPDAILGSYLLATHFDKNWYKAWHNWALANFEVISMVQEETKLNGGKND 1777 NP_004949 QLNLQGINESTIP-KVLQYYSAATEHDRSWYKAWHAWAVMNFEAVLHYKHQNQARDEKKK :: * ..* * :* * **..*:.****** **: ***.: :.:.: .. *:. P35169 DDDDTAVNNDNVRIDGSILGSGSLTING-------------NRYPLELIQR--------- 1815 NP_004949 LRHASGANITNATTAATTAATATTTASTEGSNSESEAESTENSPTPSPLQKKVTEDLSKT . :..* *. .: .:.: * . * . . :*: P35169 ---HVVPAIKGFFHSISLLETSCLQDTLRLLTLLFNFGGIKEVSQAMYEGFNLMKIENWL 1872 NP_004949 LLMYTVPAVQGFFRSISLSRGNNLQDTLRVLTLWFDYGHWPDVNEALVEGVKAIQIDTWL :.***::***:**** . . ******:*** *::* :*.:*: **.: ::*:.**

P35169 EVLPQLISRIHQPDPTVSNSLLSLLSDLGKAHPQALVYPLTVAIKSESVSRQKAALSIIE 1932 NP_004949 QVIPQLIARIDTPRPLVGRLIHQLLTDIGRYHPQALIYPLTVASKSTTTARHNAANKILK :*:****:**. * * *.. : .**:*:*: *****:****** ** :.:*::** .*:: P35169 KIRIHSPVLVNQAELVSHELIRVAVLWHELWYEGLEDASRQFFVEHNIEKMFSTLEPLHK 1992 NP_004949 NMCEHSNTLVQQAMMVSEELIRVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHA :: ** .**:** :**.******:****:*:****:*** :* *:*:: **..***** P35169 HLGNEPQTLSEVSFQKSFGRDLNDAYEWLNNYKKSKDINNLNQAWDIYYNVFRKITRQIP 2052 NP_004949 MMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQLP : . ****.*.**::::**** :* ** .:* ** ::::*.****:**:***:*::*:* P35169 QLQTLDLQHVSPQLLATHDLELAVPGTYFPGKPTIRIAKFEPLFSVISSKQRPRKFSIKG 2112 NP_004949 QLTSLELQYVSPKLLMCRDLELAVPGTYDPNQPIIRIQSIAPSLQVITSKQRPRKLTLMG ** :*:**:***:** :********** *.:* *** .: * :.**:*******::: * P35169 SDGKDYKYVLKGHEDIRQDSLVMQLFGLVNTLLKNDSECFKRHLDIQQYPAIPLSPKSGL 2172 NP_004949 SNGHEFVFLLKGHEDLRQDERVMQLFGLVNTLLANDPTSLRKNLSIQRYAVIPLSTNSGL *:*::: ::******:***. ************ **. .::::*.**:*..****.:*** P35169 LGWVPNSDTFHVLIREHRDAKKIPLNIEHWVMLQMAPDYENLTLLQKIEVFTYALDNTKG 2232 NP_004949 IGWVPHCDTLHALIRDYREKKKILLNIEHRIMLRMAPDYDHLTLMQKVEVFEHAVNNTAG :****:.**:*.***::*: *** ***** :**:*****::***:**:*** :*::** * P35169 QDLYKILWLKSRSSETWLERRTTYTRSLAVMSMTGYILGLGDRHPSNLMLDRITGKVIHI 2292 NP_004949 DDLAKLLWLKSPSSEVWFDRRTNYTRSLAVMSMVGYILGLGDRHPSNLMLDRLSGKILHI :** *:***** ***.*::***.**********.******************::**::**

P35169 DFGDCFEAAILREKYPEKVPFRLTRMLTYAMEVSGIEGSFRITCENVMRVLRDNKESLMA 2352 NP_004949 DFGDCFEVAMTREKFPEKIPFRLTRMLTNAMEVTGLDGNYRITCHTVMEVLREHKDSVMA *******.*: ***:***:********* ****:*::*.:****..**.***::*:*:** P35169 ILEAFALDPLIHWGFDLPPQKLTEQT---------GIPLPLINPSEL----LRKGAITVE 2399 NP_004949 VLEAFVYDPLLNWRLMDTNTKGNKRSRTRTDSYSAGQSVEILDGVELGEPAHKKTGTTVP :****. ***::* : . * .::: * .: ::: ** :* . ** P35169 EAAN---MEAEQQNETKNARAMLVLRRITDKLTGNDIKRFNELDVPEQVDKLIQQATSIE 2457 NP_004949 ESIHSFIGDGLVKPEALNKKAIQIINRVRDKLTGRDFSHDDTLDVPTQVELLIKQATSHE *: : :. : *: * :*: ::.*: *****.*:.: : **** **: **:**** * P35169 RLCQHYIGWCPFW-2470 NP_004949 NLCQCYIGWCPFW-2549 .*** ********

RAPTOR

FKBP12/Rpm

H E A T

FATC

LST8

RBD PIKK

In mTOR1C1 complex, eleven HEAT domains in the N-terminal part (1-1100 AAs) are followed by the FAT domain (1200-1900

AAs), small size Rpm binding followed with phosphoinositide kinase-related kinase (PIKK) domain, its regulatory domain (RD) and

C-terminal (FATC) domain. HEAT domains of RAPTOR bind to the HEAT domains of mTOR1, which makes the kinase domain to

be susceptible for inhibition with the FKBP12a/Rpm complex. The RBD binds also LST8, which probably facilitates signaling. In

Rpm-insensitive mTOR1, called mTORC2, RAPTOR is replaced with RICTOR and probably is associated with MAPKAP1 also

known as SIN1.

RD

FAT

mTORC1

1

Fig. Fs14 Alignment of two sequences: human mTOR with its homologue (NP_001175.2 also known as AAC50405.1) and their C-terminal sequences containing kinase domains; alignment-similarity score ID = 12%

AAC50405 MGEHGLELASMIPALRELGSATPEEYNTVVQKPRQILCQFIDRILTDVNVVAVELVKKTD NP_004949 MLGTGPAAATTAATTSSNVSVLQQFASGLKSRNEETRAKAAKELQHYVTMELREMSQEES * * *: .: . *. : . : .: .: .: ..: *.: *: :: . AAC50405 SQPTSVMLLDFIQHIMKSSPLMFVNVSGSHEAKGSCIEFSNWIITRLLRIAATPSCHLLH NP_004949 TR---------FYDQLNHHIFELVSSSDANERKGGILAIASLIGVEGGNATRIGRFANYL :: : . :: : :*. *.::* **. : ::. * .. . : AAC50405 KKICEVICSLLFLFKSKSPAIFGVLTKELLQLFEDLVYLHRRNVMGHAVEWPVVMSRFLS NP_004949 RNLLPSNDPVVMEMASKAIGRLAMAGDTFTAEYVEFEVKRALEWLGADRNEGRRHAAVLV ::: .::: : **: . :.: . : : :: : : :* : : .* AAC50405 QLDEHMGYLQSAPLQLMSMQNLEFIEVTLLMVLTRIIAIVFFRRQELLLWQIGCVLLEYG NP_004949 LRELAISVPTFFFQQVQPFFDNIFVAVWDPKQAIREGAVAALRACLILTTQREPKEMQKP : :. *: .: : *: * * *:. :* :* * :: AAC50405 SPKIKSLAISFLTELFQLGGLPAQPASTFFSSFLELLKHLVEMDTDQLKLYEEPLSKLIK NP_004949 QWYRHTFEEAEKGFDETLAKEKGMNRDDRIHGALLILNELVRISSMEGERLREEMEEITQ . ::: : *. . . : . * :*:.**.:.: : : .* :.:: : AAC50405 TLFPFEAEAYRNIEPVYLNMLLEKLCVMFEDGVLMRLKSDLLKAALCHLLQYFLKFVPAG NP_004949 QQLVHDKYCKDLMG-------FGTKPRHITPFTSFQAVQPQQSNALVGLLGYSSHQGLMG : .: . : : . : . :: . . ** ** * : * AAC50405 Y-ESALQVRKVYVRNICKALLDVLGIEVDAEYLLGPLYAALKMESMEIIEEIQCQTQQE- NP_004949 FGTSPSPAKSTLVESRCCRDLMEEKFDQVCQWVLKCRNSKNSLIQMTILNLLPRLAAFRP : *. .:.. *.. * * :: .:::* : .: .* *:: : : . AAC50405 NLSSNSDGISPKRRRLSSSLNPSKRAPKQTEEIKHVDMNQKSILWSALKQKAESLQISLE NP_004949 SAFTDTQYLQDTMNHVLSCVKKEKERTAAFQALGLLSVAVRSEFKVYLPRVLDIIRAALP . :::: :. . .:: *.:: .*. . : : :.: :* : * : : :: :*

2

AAC50405 YSGLKNPVIEMLEGIAVVLQLTALCTVHCSHQNMNCRTFKDCQHKSKKKPSVVITWMSLD NP_004949 PKDFAHKRQKAMQVDATVFTCISMLARAMGPG------------IQQDIKELLEPMLAVG ..: : : :: *.*: :: : . .:. .:: . :::. AAC50405 FYTKVLKSCRSLLESVQKLDLEATIDKVVKIYDALIYMQVNSSFEDHILEDLCGMLSLPW NP_004949 LSPALTAVLYDLSRQIPQLKKDIQDGLLKMLSLVLMHKPLRHPGMPKGLAHQLASPGLTT : . : .* ..: :*. : . : : .*:: :. . : * . . .*. AAC50405 IYSHSDDGCLKLTTFAANLLTLSCRISDSYSPQAQSRCVFLLTLFPRRIFLEWRTAVYNW NP_004949 LPEASDVGSITLALRTLGSFEFEG---HSLTQFVRHCADHFLNSEHKEIRMEAARTCSRL : . ** *.:.*: : . : :. .* : .: . .:*. :.* :* : . AAC50405 ALQSSHEVIRASCVSGFFILLQQQNSCNRVPKILIDKVKDDS-DIVKKEFASILGQLVCT NP_004949 LTPSIHLISGHAHVVSQTAVQVVADVLSKLLVVGITDPDPDIRYCVLASLDERFDAHLAQ * * : : * . : : .:: : * . . * * .: . :. :. AAC50405 LHGMFYLTSSLTEPFSEHGHVDLFCRNLKATSQHECSSSQLKASVCKPFLFLLKKKIPSP NP_004949 AENLQALFVALNDQVFEIRELAICTVGRLSSMNPAFVMPFLRKMLIQILTELEHSGIG-R ..: * :*.: . * .: : . :: : . *: : : : * :. * AAC50405 VKLAFIDNLHHLCKHLDFREDETDVKAVLGTLLNLMEDPDKDVRVAFSGNIKHILESLDS NP_004949 IKEQSARMLGHLVSNAPRLIR-PYMEPILKALILKLKDPDPDPNPGVINNVLATIGELAQ :* * ** .: . ::.:* :*: ::*** * . .. .*: : .* . AAC50405 ED-----GFIKELFVLRMKEAYTHAQISRNNELKDTLILTTGDIGRAAKGDLVPFALLHL NP_004949 VSGLEMRKWVDELFIIIMDMLQDSSLLAKRQVALWTLGQLVASTGYVVEPYRKYPTLLEV . ::.***:: *. : :::.: ** ... * ..: :**.: AAC50405 LHCLLSKSASVSGAAYTEIRALVAAKSVKLQSFFSQYKKPICQFLVESLHSSQMTALPNT NP_004949 LLNFLKTEQNQG-------TRREAIRVLGLLGALDPYKHKVNIGMIDQSRDASAVSLSES * :*... . . * : : * . :. **: : :::. :.:. .:*.:: AAC50405 PCQNADVRKQDVAHQREMALNTLSEIANVFDFPDLNRFLTRTLQVLLPDLAAKASPAASA NP_004949 KSSQDSSDYSTSEMLVNMGNLPLDEFYPAVSMVALMRIFR---DQSLSHHHTMVVQAITF ..: . . :*. .*.*: ...: * *:: : *.. : . * : AAC50405 LIRTLGKQLNVNRREILINNFKYIFSHLVCSCSKDELERALHYLKNETEIELGSLLRQDF NP_004949 IFKSLGLKCVQFLPQVMPTFLNVIR--VCDGAIREFLFQQLGMLVSFVKSHIRPYMDEIV ::::** : ::: . :: * : .. :: * : * * . .: .: . : : .

3

AAC50405 QGLHNELLLRIGEHYQQVFNGLSILASFASSDDPYQGPRDIISPELMADYLQPKLLGILA NP_004949 TLMREFWVMNTSIQSTIILLIEQIVVALGGEFKLYLP---QLIPHMLRVFMHDNSPGRIV ::: ::. . : :: .*:.::... . * : *.:: ::: : * :. AAC50405 FFNMQLLSSSVGIEDKKMALNSLMSLMKLMGPKHVSSVRVKMMTTLRTGLRFKDDFPELC NP_004949 SIKLLAAIQLFGANLDDYLHLLLPPIVKLFDAPEAPLPSRKAALETVDRLTESLDFTDYA ::: . .* : .. * .::**:.. ... * * . **.: . AAC50405 CRAWDCFVRCLDH-ACLGSLLSHVIVALLPLIHIQPKETAAIFHYLIIENRDAVQDFLHE NP_004949 SRIIHPIVRTLDQSPELRSTAMDTLSSLVFQLGKKYQIFIPMVNKVLVRHRINHQRYDVL .* . :** **: . * * ..: :*: : : : .:.: :::.:* * : AAC50405 IYFLPDHPELKKIKAVLQEYRKETSESTDLQTTLQLSMKAIQHENVDVRIHALTSLKETL NP_004949 ICRIVKGYTLADEEEDPLIYQHRMLRSGQGDALASGPVETGPMKKLHVSTINLQKAWG-- * : . * . : *::. .* : :: . .::: :::.* * . AAC50405 YKNQEKLIKYATDSETVEPIISQLVTVLLKGCQDANSQARLLCGECLGELGAIDPGRLDF NP_004949 ----------AARRVSKDDWLEWLRRLSLELLKDSSSPSLRSCWALAQAYNPMARDLFNA *: : : :. * : *: :*:.* : * ..: . :: AAC50405 STTETQGKDFTFVTGVEDSSFAYGLLMELTRAYLAYADNSRAQDSAAYAIQELLSIYDCR NP_004949 AFVSCWS----------------ELNEDQQDELIRSIELALTSQDIAEVTQTLLNLAEFM : .. . * : : : : :.:. * . * **.: : AAC50405 EMETNGPGHQLWRRFPEHVREILEPHLNTRYKSSQKSTDWSGVKKPIYLSKLGSNFAEWS NP_004949 EHSDKGP--------------LPLRDDNGIVLLGERAAKCRAYAKALHYKELEFQKGPTP * . :** : . * .::::. . *.:: .:* : . . AAC50405 ASWAGYLITKVRHDLASKIFTCCSIMMKHDFKVTIYLLPHILVYVLLGCNQEDQQEVYAE NP_004949 AILESLISINNKLQQPEAAAGVLEYAMKH--------------FGELEIQATWYEKLHEW * . : : : : .. . *** : * : :::: AAC50405 IMAVLKHDDQHTINTQDIASDLCQLSTQTVFSMLDHLTQWARHKFQALKAEKCPHSKSNR NP_004949 EDALVAYDKKMDTNKDDPELMLGRMRCLEALGEWGQLHQQCCEKWTLVNDETQAKMAR-- *:: :*.: *.:* * :: .:. .:* * . .*: :: *. .: AAC50405 NKVDSMVSTVDYEDYQSVTRFLDLIPQDTLAVASFRSKAYTRAVMHFESFITEKKQNIQE NP_004949 ---MAAAAAWGLGQWDSMEEYTCMIPRDTHDGAFYR-----AVLALHQDLFSLAQQCIDK : .:: . :::*: .: :**:** * :* .: .:.::: :* *::

4

AAC50405 HLGFLQKLYAAMHEPDGVAGVSAIRKAEPSLKEQILEHESLGLLRDATACYDRAIQLEPD NP_004949 ARDLLDAELTAMAGESYSRAYGAMVSCH--------MLSELEEVIQYKLVPERREIIRQI .:*: :** . . .*: ... ..* : : . :* :. AAC50405 QIIHYHGVVKSMLGLGQLSTVITQVNGVHANRSEWTDELNTYRVEAAWKLSQWDLVENYL NP_004949 WWERLQGCQRIVEDWQKILMVRSLVVSPHEDMRTWLKYASLCGKSGRLALAHKTLVLLLG : :* : : . :: * : * . * : * . . .. *:: ** AAC50405 AADGKSTTWSVRLGQLLLS--------AKKRDITAFYDSLKLVRAEQIVPLSAASFERGS NP_004949 VDPSRQLDHPLPTVHPQVTYAYMKNMWKSARKIDAFQHMQHFVQTMQQQAQHAIATED-- . .:. .: : :: . *.* ** . ::*:: * . * : * AAC50405 YQRGYEYIVRLHMLCELEHSIKPLFQHSPGDSSQEDSLNWVARLEMTQNSYRAKEPILAL NP_004949 -QQHKQELHKLMARCFLKLGEWQLNLQGINESTIPKVLQYYSAATEHDRSWYKAWHAWAV *: : : :* * *: . * :. .:*: . *:: : :.*: *: AAC50405 RRALLSLNKRPDYNEMVGECWLQSARVARKAGHHQTAYNALLNAGESRLAELYVERAKWL NP_004949 MNFEAVLHYKHQN----------QARDEKKKLRHASGANITN---ATTAATTAATATTTA . *: : : .** :* :* :. * : * . :. AAC50405 WSKGDVHQALIVLQKGVELCFPENETPPEGKNMLIHGRAMLLVGRFMEETANFESNAIMK NP_004949 STEGSNSESEAESTENSPTPSPLQKKVTEDLSKTLLMYTVPAVQGFFRSISLSRG----N ::*. :: :. * ::. .*. . : :: * *:.. : .. : AAC50405 KYKDVTACLPEWEDGHFYLAKYYDKLMPMVTDNKMEKQGDLIRYIVLHFGRSLQYGNQFI NP_004949 NLQDTLRVLTLWFD-YGHWPDVNEALVEGVKAIQIDTWLQVIPQLIARIDTPRPLVGRLI : :*. *. * * : : .. : *: *. :::. ::* :: ::. . .::* AAC50405 YQSMPRMLTLWLDYGTKAYEWEKAGRSDRVQMRNDLGKINKVITEHTNYLAPYQFLTAFS NP_004949 HQLLTDIG--RYHPQALIYPLTVASKSTTTARHNAANKILKNMCEHSNTLVQQAMMVS-E :* :. : . : * *.:* . :* .** * : **:* *. ::.: . AAC50405 QLISRICHSHDEVFVVLMEIIAKVFLAYPQQAMWMMTAVSKSSYPMRVNRCKEILNKAIH NP_004949 ELIRVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS----- :** *: . * * * :.*: : :: : ** AAC50405 MKKSLEKFVGDATRLTDKLLELCNKPVDGSSSTLSMSTHFKMLKKLVEEATFSEILIPLQ NP_004949 FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQLPQLTSLELQYVSPK :::: : : :* . * :: * .: * .. :: *:* : :::. :. :

5

AAC50405 SVMIPTLPSILGTHANHASHEPFPGHWAYIAGFDDMVEILASLQKPKKISLKGSDGKFYI NP_004949 LLMCRDLELAVPGTYDPN------QPIIRIQSIAPSLQVITSKQRPRKLTLMGSNGHEFV :* * : : * .: :::::* *:*:*::* **:*: :: AAC50405 MMCKPKDDLRKDCRLMEFNSLINKCLRKDAESRRRELHIRTYAVIPLNDECGIIEWVNNT NP_004949 FLLKGHEDLRQDERVMQLFGLVNTLLANDPTSLRKNLSIQRYAVIPLSTNSGLIGWVPHC :: * ::***:* *:*:: .*:*. * :*. * *::* *: ******. :.*:* ** : AAC50405 AGLRPILTKLYKEKGVYMTGKELRQCMLP---KSAALSEKLKVFREFLLPRHPPIFHEWF NP_004949 DTLHALIRDYREKKKILLNIEHRIMLRMAPDYDHLTLMQKVEVFEHAVNNTAGDDLAKLL *:.:: . ::* : :. :. :. . :* :*::**.. : : : : AAC50405 LRTFPDPTSWYSSRSAYCRSTAVMSMVGYILGLGDRHGENILFDSLTGECVHVDFNCLFN NP_004949 WLKSPSSEVWFDRRTNYTRSLAVMSMVGYILGLGDRHPSNLMLDRLSGKILHIDFGDCFE . *.. *:. *: * ** **************** .*:::* *:*: :*:**. *: AAC50405 KGET-FEVPEIVPFRLTHNMVNGMGPMGTEGLFRRACEVTMRLMRDQREPLMSVLKTFLH NP_004949 VAMTREKFPEKIPFRLTRMLTNAMEVTGLDGNYRITCHTVMEVLREHKDSVMAVLEAFVY . * :.** :*****: :.*.* * :* :* :*...*.::*::::.:*:**::*:: AAC50405 DPLVEWSKPVKG-----------------------------------------HSKAPLN NP_004949 DPLLNWRLMDTNTKGNKRSRTRTDSYSAGQSVEILDGVELGEPAHKKTGTTVPESIHSFI ***::* .. .* .: AAC50405 ETGEVVNEKAKTHVLDIEQRLQGVIKTRNRVTGLPLSIEGHVHYLIQEATDENLLCQMYL NP_004949 GDGLVKPEALNKKAIQIINRVRDKLTGRDFSHDDTLDVPTQVELLIKQATSHENLCQCYI * * * :.:.::* :*::. :. *: . .*.: :*. **::**..: *** *: AAC50405 GWTPYM NP_004949 GWCPFW ** *:

Red – FKBD in human mTOR Cyan – pre-kinase sequence Green – kinase domain

6

Alignment of C-terminus containing kinase domain marked in pink; sequence similarity is ID = 29% AAC50405 SVMIPTLPSILGTHANHASHEPFPGHWAYIAGFDDMVEILASLQKPKKISLKGSDGKFYI NP_004949 -------------------------------------------------------GHEFV *: :: AAC50405 MMCKPKDDLRKDCRLMEFNSLINKCLRKDAESRRRELHIRTYAVIPLNDECGIIEWVNNT NP_004949 FLLKGHEDLRQDERVMQLFGLVNTLLANDPTSLRKNLSIQRYAVIPLSTNSGLIGWVPHC :: * ::***:* *:*:: .*:*. * :*. * *::* *: ******. :.*:* ** : AAC50405 AGLRPILTKLYKEKGVYMTGKELRQCMLP---KSAALSEKLKVFREFLLPRHPPIFHEWF NP_004949 DTLHALIRDYREKKKILLNIEHRIMLRMAPDYDHLTLMQKVEVFEHAVNNTAGDDLAKLL *:.:: . ::* : :. :. :. . :* :*::**.. : : : : AAC50405 LRTFPDPTSWYSSRSAYCRSTAVMSMVGYILGLGDRHGENILFDSLTGECVHVDFNCLFN NP_004949 WLKSPSSEVWFDRRTNYTRSLAVMSMVGYILGLGDRHPSNLMLDRLSGKILHIDFGDCFE . *.. *:. *: * ** **************** .*:::* *:*: :*:**. *: AAC50405 KGET-FEVPEIVPFRLTHNMVNGMGPMGTEGLFRRACEVTMRLMRDQREPLMSVLKTFLH NP_004949 VAMTREKFPEKIPFRLTRMLTNAMEVTGLDGNYRITCHTVMEVLREHKDSVMAVLEAFVY . * :.** :*****: :.*.* * :* :* :*...*.::*::::.:*:**::*:: AAC50405 DPLVEWSKPVK-----------------GHSKAPLN--ETGEVVNEKAKTHVLDIEQRLQ NP_004949 DPLLNWRLMDTNTKGNKRSRTRTDSYSAGQSVEILDGVELGEPAHKKTGTTVPESIHSFI ***::* . *:* *: * ** .::*: * * : : : AAC50405 G----------------VIKTRNRVTG------LPLSIEGHVHYLIQEATDENLLCQMYL NP_004949 GDGLVKPEALNKKAIQIINRVRDKLTGRDFSHDDTLDVPTQVELLIKQATSHENLCQCYI * : :.*:::** .*.: :*. **::**..: *** *: AAC50405 GWTPYM NP_004949 GWCPFW ** *:

Table Ts1. Numbers of inter-molecular interactions calculated at distance ! 4.5 Å from 1TOC.pdb; CnA – calcineurin A subunit; CnB calcineurin B subunit; FKBP12 – immunophilin. CnA/FK506 CnB/FK506 FKBP12/FK506 FKBP12/CnA FKBP12/CnB CnA/CnB TRP352 15 MET118 3 TYR26 8 THR27 1 ARG42 18 VAL21 4 SER353 4 VAL119 4 PHE36 4 ASP32 5 ASN43 2 PHE23 10 PRO355 2 ASP37 11 LYS34 8 LYS44 12 PRO24 3 PHE356 14 ARG42 3 LYS35 5 PRO45 2 GLU53 6 GLU359 2 PHE46 11 PHE36 3 PHE46 9 ARG55 3 GLU54 3 ASP37 4 LYS47 3 ILE168 2 VAL55 5 SER38 2 GLN53 4 PRO340 6 ILE56 6 ARG40 4 GLU54 5 TYR341 15 TRP59 15 ASP41 5 TRP342 10 ALA81 2 ARG42 7 LEU343 3 TYR82 20 HIS87 3 ASP348 8 HIS87 8 PRO88 8 VAL349 14 ILE91 2 GLY89 3 PHE350 11 PHE99 5 ILE90 5 THR351 2 TRP352 4 SER353 8 LEU354 8 PHE356 6 VAL357 7 LYS360 13 VAL361 6 THR362 4 GLU363 2 MET364 17 LEU365 6 ASN367 14 VAL368 10 LEU369 16 ILE371 11 CYS372 2