Crystal Structure of Borrelia turicatae protein, BTA121, a differentially regulated gene in the tick-mammalian transmission cycle of relapsing fever spirochetes

Supplementary Information for Crystal Structure of Borrelia turicatae protein, BTA121, a

differentially regulated gene in the tick-mammalian transmission cycle of relapsing fever

spirochetes.

Zhipu Luo

, Alan J. Kelleher

, Rabih Darwiche

, Elissa H. Hudspeth

,

Oluwatosin K. Shittu

,

Aparna Krishnavajhala

, Roger Schneiter

, Job E. Lopez

and Oluwatoyin A. Asojo

1

_{Synchrotron Radiation Research Section, Macromolecular Crystallography Laboratory,}

National Cancer Institute, Argonne, Illinois 60439, USA

2

_{National School of Tropical Medicine, Baylor College of Medicine, Houston Texas, United}

States of America

3

_{Division of Biochemistry, Department of Biology, University of Fribourg, Chemin du Musée}

10, CH 1700 Fribourg, Switzerland

Running Title: Crystal Structure of BTA121

Keywords: relapsing fever; neglected tropical diseases; vector adaptation; lipid binding; tick

borne diseases; structure-function relationships; bioinformatics; argasids ticks

Purification of BTA121

A B

Figure S.1: Confirmation of cleavage of Histag by anti-His Western blotting. Lane A contains

BTA121-His while Lane B contains BTA121 post removal of the Histag.

Figure S2: A) SDS-page gel showing purity of BTA121, visualized by Coomassie blue staining.

Electrophoretic mobility on the reduced gel is consistent with the theoretical molecular mass for

the tagged and untagged proteins. B) 3D gel of Fig S.2A visualized by false color.

Size exclusion chromatography

The molecular mass of the eluted peak is determined using a calibration curve based on known

standards, not just by visualization. The standard consisted of bovine thyroglobulin (670 kDa),

bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and

vitamin B12 (1.35 kDa), which was injected onto the column using the same method as the

BTA121 samples. For samples analyzed on different days or with different column/guard

cartridge set-ups, a gel filtration standard was injected on the same day and with the same set-up

as the experimental sample. In addition, blank injections of the mobile phases were used to

confirm a stable baseline and the absence of carry-over (prior injected sample eluting during the

subsequent injection run). Blue dextran was injected to estimate the retention time of the void

volume (the volume present from the injection loop to the PDA detector, excluding the pore

volume of the beads). This provides the retention time at which a compound will elute that is

larger than the pore size of the beads (145 µm diameter) and has no interaction with the bead

surface. A 1 M sodium chloride (NaCl) solution was injected to estimate the pore volume of the

beads. The retention times of the protein standards and blue dextran were utilized to calculate

the distribution coefficient (Kd) for each standard and to create a plot of the log of the molecular

weight (MW) versus Kd. Normalizing the retention times to Kd removes system and column

specific information (void and column volume) allowing direct comparison of the data to that

obtained from other systems and columns. The slope and y-intercept of a best-fit linear line was

used to calculate the molecular weight of the sample peaks.

Kd (distribution coefficient) = (Ve - Vo) / (Vt - Vo)

Ve = elution volume of sample

Vo = void volume (blue dextran)

250

200

150

100

50

0

670

158

44

17

1.35

mA

U

Figure S.3 Purity of freshly thawed untagged BTA121 reveals two peaks. These can be separated

by gel filtration, however removing the minor peak does not improve the quality of the crystals.

Similar profile was observed for the selenomethionine protein.

Figure S.4: Extensive peak broadening occurs after storing BTA121 at 4

C. The sample stored at

28 mg/mL for greater than 70 days (red) has a consistent profile with the sample stored at 5

mg/mL for 43 days (blue). Since 100 µg was injected of the sample stored at 28 mg/mL and 50

µg was injected of the sample stored at 5 mg/mL, the chromatograph of the 28 mg/mL sample

was adjusted 2-fold for comparison.

Figure S.5. Structural and primary sequence alignment of BTA121 with other B. turicatae

proteins from the megaplasmid. This figure was generated with ESPript

. The different

secondary structure elements shown are alpha helices as large squiggles labelled (a), 310-helices

as small squiggles labelled (h), beta strands as arrows (b), and beta turns (TT). Identical residues

are shown in white on red background, and conserved residues in red. The locations of the

cysteine residues involved in disulfide bonds are numbered in green.

1

Gouet, P., Robert, X. & Courcelle, E. ESPript/ENDscript: Extracting and rendering sequence and 3D

information from atomic structures of proteins. Nucleic Acids Res 31, 3320-3323 (2003).

fBTA121

1 1 0 2 0 3 0 4 0 5 0 6 0 X X X X X X X

fBTA121 DEEEF DEEEEEEF DEEF DEEF DEF A M L K I K F S L L L V L L L L L I S C D S K A T C T L V S G V A G D T E T E A V T N T G A G L E A G V R P G V G L R I S

BTA121 KdddG KddddddG KddG KddG KdG B . . . . BTA121-His KdddG KddddddG KddG KddG KdG B . . . H H H H H H H H BTA116 KdddG KddddddG KddG KddG KdG B M L K I K F S L L L I L I L L L L I S C D K A T C L F L G I R T N R A . . . . BTA123 KdddG KddddddG KddG KddG KdG B M L K I K F S L L L V L I L L L L I S C D S K A T C L F S G G A G N T G A G G G A G S G . . T N T . . . . BTA126 KdddG KddddddG KddG KddG KdG B M V K I K F S V L L V L I L L L L I S C D S K A T C L V S G G A G N T R A V V K S V A G . . T N T R A V V K S . . . . . BTA127 KdddG KddddddG KddG KddG KdG B M L I R S V L L V L I L L L I N C D S K A T N H I F F F S R G A G N T G A G G G A G . . . . BTA119 KdddG KddddddG KddG KddG KdG B M V K I K F S L L L V L I L L L L I S C D S K A T C L V S G G A G N T G I T T G K T T K . . I T T G T N T . . . . BTA118 KdddG KddddddG KddG KddG KdG B M V K I K F S L L L V L I L L L L I S C D S K A T C L F S G G A G N T G I T T G K T T K . . I T T G T T T G T . . . . . BTA117 KdddG KddddddG KddG KddG KdG B M L K I K F S L L L V L I L L L L I S C D S K A T C L V S G G A G N T V T D T G K T T K . . I T T G T T T G T . . . . . BTA122 KdddG KddddddG KddG KddG KdG B M L K I K F S L L L V L I L L L L I S C D S K A T C L F S G G A G . . . . BTA124 KdddG KddddddG KddG KddG KdG B M V K I K F S L L L V L I L L L L I S C S K A T C F V G S G V T N . . . G K T T G . . T N T G I T T G T . . . . . BTA120 KdddG KddddddG KddG KddG KdG B M L K I K F S L L L V L I L L L L I S C D S K A T C L V S G G A G N T V T D T G K T T G . . T N T G . . . . BTA115 KdddG KddddddG KddG KddG KdG B M V K I K F S L L L I L I L L L L I S C D K A T C L F S G I G T N K T E V K P G A G . . . . L D A V V K P G A G L D A V BTA112 KdddG KddddddG KddG KddG KdG B M L K V R N L L V L I L L L L M C S Y R S L V G N F K P K D A A A S G E N S I V K K N E . . . .

BTA129 JIIIH JIIIIIIH JIIH JIIH JIH C M M K L F S L L V L I I L L L V N C D S K S H F F V L K G S T G I E E N L . T S K R E V K K . . . .

fBTA121 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 X X X X X X fBTA121 T V P V V V I R L E S G V R P E S G V G P E S G V G P E A G V G P E A G V G P E S G V G P E A G V G P E A G V G P E S G BTA121 . . . G G V R P E S G V G P E S G V G P E A G V G P E A G V G P E S G V G P E A G V G P E A G V G P E S G BTA121-His H H H H E N L Y F Q G G V R P E S G V G P E S G V G P E A G V G P E A G V G P E S G V G P E A G V G P E A G V G P E S G BTA116 . . . . BTA123 . . . . BTA126 . . . G S G T N T R . . . T V V E BTA127 . . . . BTA119 . . . . BTA118 . . . T T G T T T . . . . BTA117 . . . N T G T N T G T N T G T N T G T T T G T N . . . T G T N T G . . T N T G BTA122 . . . . BTA124 . . . N T G . . I T T G BTA120 . . . . BTA115 V V . . . K P . . . G A G L E A G A G L G A G E . . . A G L G . A G G A G L E A G E A G E A G . . . . BTA112 . . . . BTA129 . . . L G . . . . iiiiii fBTA121 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 X X X X X X

fBTA121 c A DEEEF DEEF A A DEF D L E V G D E E L A Y L E V P K L V G P E S G V G P E A G V R P E A G V R P E A G A G T D T E T E E E I E V A N T I D I A

BTA121 c B KdddG KddG B B KdG K L E V G D E E L A Y L E V P K L V G P E S G V G P E A G V R P E A G V R P E A G A G T D T E T E E E I E V A N T I D I A BTA121-His c B KdddG KddG B B KdG K L E V G D E E L A Y L E V P K L V G P E S G V G P E A G V R P E A G V R P E A G A G T D T E T E E E I E V A N T I D I A BTA116 c B KdddG KddG B B KdG K L E V G D E E L A Y L D P K L . . . E L R T K A E I E D I . S D K A BTA123 c B KdddG KddG B B KdG K L L D E D L A Y L D V P L . . . G V G A G G G A E K E E K I D E M A BTA126 c B KdddG KddG B B KdG K L D V D E D L A Y L E V P R S G S D K E L G . . . A V T G I D E R T E P K P E E E T G L I T V E K E N T I D F Q A BTA127 c B KdddG KddG B B KdG K L E V G D E E L A Y L E V K L . . . S G I N T R P E P N T V E A E K T D S K A BTA119 c B KdddG KddG B B KdG K L D V G D E E L A Y L E V P K L . . . G T N T G T N T V T V E A N T T Y K E BTA118 c B KdddG KddG B B KdG K L D V G D E E L A Y L E V P K L . . . G . . . T N T G I T T E T N T G T T T G T N T V T V E A N T T Y K E BTA117 c B KdddG KddG B B KdG K L D V G D E E L A Y L D V P K L T N T G T N T G . . . T N T G T N T G T N T G T N T G T N T V T V E A E K T Y I A BTA122 c B KdddG KddG B B KdG K L E V G D E E L A Y L D V P K L . . . D T T E A E K T Y K E BTA124 c B KdddG KddG B B KdG K L D V G D E E L A Y L V P R V T N T E I T T G . . . T N T E I T T G T N T G T N T G T N T G T V E G E G K T D Q A BTA120 c B KdddG KddG B B KdG K L E V G D E E L A Y L D K L . . . T N T G . . T D T A E A E I . S D S K E BTA115 c B KdddG KddG B B KdG K L A G E A L D I P R L . . . E A G L E A . . . G E A G L E A G . . . . L E A G E A G L E A G E L A G E G . D K S K A BTA112 c B KdddG KddG B B KdG K L D K L . . . K P K E H . . . D T E S L N A D N

BTA129 c C JIIIH JIIH C C JIH J L V K L . . . R F G . . . R E Y T S K M A . . . T G N N L D I N N

iiii iiiiiiiiiii i iiiiiiii iiiiiii iiii

. . . fBTA121

1 9 0 2 0 0 2 1 0 2 2 0 X X X X

fBTA121 c c c c c cc EF DEEF DEEEF DF A A A DF DEEEEEEEEEEEEEL I L L A I I D F G V S G R K A I Y L D Y N G D V V I E K K P T D R S S Q G N T S . . . V I D R K

BTA121 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y L D Y N G D V V I E K K P T D R S S Q G N T S . . . V I D R K BTA121-His c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y L D Y N G D V V I E K K P T D R S S Q G N T S . . . V I D R K BTA116 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A V Y L D Y N G D V V I E K K P T Q G A I R G G . . . L G D V D R K BTA123 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y N G D V V I E K K P T Q G P G G G K F R D S . . . G D V H K R A BTA126 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G K K I Y L Y N G D V I E K K P T N G A T S Q R T P A . . . N V V D Y Q T BTA127 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y L D Y N G D V V I E K P T Q E A G Q G I R V S . . . Y K N G F D K F N C Q E BTA119 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y L D Y N G D V A V E K K P T Q G A S Q G I S V S . . . Y K D G F D K F N C K BTA118 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y L D Y N G D V A V E K K P T Q G A S Q G I R V S . . . Y K N G F D K F N S Q BTA117 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y L D Y N G D V V V E K K P T D G A G Q E I S F S . . . Y V D E F D K F N S K BTA122 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D G V S G R K A I Y L D Y N G D V V I E K K P T Q L G A G Q G I S V S . . . Y K D G F D K F N C Q BTA124 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D G V S G R K A I Y L D Y N D I V I E K K P T K L G A V R D N T G . . . D D V D F F N R K R BTA120 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F G V S G R K A I Y L D Y N G D V V I E K K P T Q G A G Q E K S V S . . . Y V D E F D K F N Y K BTA115 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I D F V S G K K A I Y D Y N D V V L K K P T D N I E G R K I F I D . . . K N N D I E K L K H S E A BTA112 c c c c c cc dG KddG KdddG KG B B B KG KdddddddddddddL I L L A I I F G I S G K R I V Y N G I K N T E A S V R K D V T D V G I G S V E G Y R N Y T D S E L A L D A K . Q E A D

BTA129 c c c c c cc IH JIIH JIIIH JH C C C JH JIIIIIIIIIIIIIL I L L A I I D F G L R A I Y I Y G I K L D K E E A R R T T D S K I G N A S Y Y K T Y T D F E F N L E S T R . K E T F Y α1

iiiiiiiiiiiiiii llliiiiiiiiiiiiiiiiiiiiiii iii iiiiii

. . . . fBTA121

2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 X X X X X X

fBTA121 EEEEEEEF DEEEF A DEEEEF A A DF A A V S L L K K G A L K I I T V K T R L Q M D L V D F T E E D P N E G N C K R Y G K G I G Y . . . . D F F H G S I G T R A Q

BTA121 dddddddG KdddG B KddddG B B KG B B V S L L K K G A L K I I T V K T R L Q M D L V D F T E E D P N E G N C K R Y G K G I G Y . . . . D F F H G S I G T R A Q BTA121-His dddddddG KdddG B KddddG B B KG B B V S L L K K G A L K I I T V K T R L Q M D L V D F T E E D P N E G N C K R Y G K G I G Y . . . . D F F H G S I G T R A Q BTA116 dddddddG KdddG B KddddG B B KG B B V S L L K A R G A L R V I T V K R L Q V D L V D F E Q D P N E G V D F H R Y N . D I R G . . . . K F I Y V V G S E N A K BTA123 dddddddG KdddG B KddddG B B KG B B V S L L K A R G A L K V T I K T R L Q M D L V D F E K E D P N E S D L N R Y T . E S V W H D . . F F H N R L I E E M V E BTA126 dddddddG KdddG B KddddG B B KG B B V S L L R A R G A L R V I T I K T L Q M D L F K E G T D E G G D F E K Y A S F S Y L W E V . . . . F N R A I K E N D V E BTA127 dddddddG KdddG B KddddG B B KG B B V S L L R A R G A L R V I T V K T R L Q M D V I D F E E G T D E G D L S S C N . S F A W N P . . D R D H L I F N E K A N BTA119 dddddddG KdddG B KddddG B B KG B B V S L L A R G A L R V I T V K S R L Q M D V I D F I K Q D P N A G D L S S C H . S L T W Y L I S G F D Y F I F E E N A K BTA118 dddddddG KdddG B KddddG B B KG B B V S L L R A R G A L R V I T V K S R L Q M D V I D F E Q D P N A G D L S S C H . S L S W Y L I S G F D Y F I F K E V T R BTA117 dddddddG KdddG B KddddG B B KG B B V S L L R A R G A L R V M T V K T R Q M D V V D F K Q G T D A G F D L S S C H . S F T W Y L . . D F D H L S F R E I A D BTA122 dddddddG KdddG B KddddG B B KG B B V S L L R A R G A L R V M T V K T R L Q M D V D F K Q G T D A G D L S S C L S . S L A W Y L . . N S N P L I F H E G A D BTA124 dddddddG KdddG B KddddG B B KG B B V L L R A R G A L R V I T L K S K L V D L V D F I E E G T D E D R L F H R Y S . . . N I R E K F N I L L L E R I E K BTA120 dddddddG KdddG B KddddG B B KG B B V S L L A R G A L R V I T V K T R L Q M D V V D F I K Q G I D A D D L S S C Y . S F T W Y V . . N F N P L I F K E V T R BTA115 dddddddG KdddG B KddddG B B KG B B V S L L R A R G A L R V I T I K S R L Q V D L V D E Q D P N E S D Y D R Y A K R E F K . . . R Y F V D R F G L I S V V D BTA112 dddddddG KdddG B KddddG B B KG B B L L V K A L A I R Q L Q F E T Q K T E N N V H K P K E N L R F N S K R N E Y . . . P L H . . . L K G L F N E S D N V

BTA129 IIIIIIIH JIIIH C JIIIIH C C JH C C L V V I R V I R K L Q D F E N Q K S F E K N V K D A T S G N E L N E R K N Q Y . . . Q L H . . . L K G L F D S N D D

i illliiiii iiiiii lllll lll iiiiiiiiiiiiii

. . . fBTA121

2 9 0 3 0 0 3 1 0 3 2 0 X X X X

fBTA121 c c c c c A A A DF DEEEEEEEEEF DEF DEEEEF A DEEF DEEEF K D Y L V A V Y R F K F E I V K N P R V I W D A D D A I E I K I I N . . . V Y A P R M D G R T N G A . . . .

BTA121 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V A V Y R F K F E I V K N P R V I W D A D D A I E I K I I N . . . V Y A P R M D G R T N G A . . . . BTA121-His c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V A V Y R F K F E I V K N P R V I W D A D D A I E I K I I N . . . V Y A P R M D G R T N G A . . . . BTA116 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V A V Y K F K F E M V K N P R V V W D D A I D E I I I S . . . T R A S S M . A D A Q T E N D . . . . BTA123 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V V Y K F K F E M V K P R V V W D A D D V I D E I K I V N S . . . T Y V P S H I . A C I E A . . . . BTA126 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V I R F K E M V S P R V V W D A D D V I D D I K I I S F . . . A R C D S R L N K . A R T G G . . . . BTA127 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V A V Y K F K F E M V K N P R V I W D A D E A I D E I I I S . . . N R T P S K . V Q T G N T . . . . BTA119 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V A V Y K F K F E M V K N P R V I W D A D E A I D D M K I V N . . . T R T P S M . V Q I E A . . . . BTA118 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V M I Y K F K F E M V K N P R V I W D A D E A I D D M K I V N . . . T R T P S M . V Q I E A . . . . BTA117 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V L I Y K F K F E M V K N P R V I W D A D E A I D E I K I V N . . . T R T S S M . V Q I E A . . . . BTA122 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V L I Y K F K F E M V K N P R V M W D A D E A I D E I K I V N . . . T R T S S K . A Q A E A . . . . BTA124 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V V Y K F K F E M V N P R L M W D A D E A I D E I K V V N T . . . T R A P R T M . A R T E F . . . . BTA120 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V I I Y K F K F E M V K N P R V V W D A D E A I D E I K I V N . . . T R T S R M . V Q A E A . . . . BTA115 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V V K F K F E M V K S P R V V W D V D E A I E I K I V N T . . . T S C T S R M . G Q T N E A . . . . BTA112 c c c c c B B B KG KdddddddddG KdG KddddG B KddG KdddG K D Y L V V Y F E I Q L E V D I V N Y S N V I G D N V N E T A I E I V I G D F K G S S R K S V Y Q S T A N F C Y A N N D

BTA129 c c c c c C C C JH JIIIIIIIIIH JIH JIIIIH C JIIH JIIIH K D Y L V I Y K F D I V W D E I I D I I Y N E I I G D N F S E L I E T K V E G G L T S D Q V K R G T D P L I G . K Y K G

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. . . . fBTA121

3 3 0 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 X X X X X X

fBTA121 cc c c DEEEEEEF DEEF A DF DF DF DF DEF A DF DEEEF DF A T Y F L D K D K N N S V V V M I Y R V K D E A L A I V D V K Q L . . N V G V Y Y R D I G I E H . . . . N T T P G V D Q A

BTA121 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K D K N N S V V V M I Y R V K D E A L A I V D V K Q L . . N V G V Y Y R D I G I E H . . . . N T T P G V D Q A BTA121-His cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K D K N N S V V V M I Y R V K D E A L A I V D V K Q L . . N V G V Y Y R D I G I E H . . . . N T T P G V D Q A BTA116 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K D K N N S L D I V I I Y R I K E E A L A I V S D D A K Q L . . S M D Y R R D I D I K Q . . . . K E G E N K T BTA123 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D D R N N S L D V I I I Y R I K E A L A I I S D D I K Q . . Q N M G A N A K I D I E Q G . . . . K D S V A Y Q D BTA126 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K D R N N L D V I I V I K E E A L A I V S D D A K Q L . . N M L E P L K V L C Q S K R Q . . . . K E G E K Q A BTA127 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K D K N N S V D V V I I R V K E E A L A I V S D D A K Q L . . N V S Y H K S F C L K M Q . . . . N E G E K Q V BTA119 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K E K N N S L D V A I V Y R I K E E A L A I V S D D A K Q L . . D K S Y R E Y N D N G R . . . . K D G E N K T BTA118 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K E K N N S L D V A I V Y R K E E A L A I V S D D A K Q L . . D K S Y R E C Y D F N D Q . . . . K E G E N K T BTA117 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K D K N N S L D V A I V Y R V K E E A L A I V S D D A K Q L . . K K S Y H T Y N D I K R . . . . K D G E N K T BTA122 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D R D K N N S L D V A I V Y R I K E E A L A V V S D D K Q L . . K K S Y Y A Y S D N D Q . . . . K E G T E N K A BTA124 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L K E K N L I V I I Y I K E E V A I V S D D R Q L . . T D D I D S S G Y K E I E D I K Q . . . . S K A T F L D Q A BTA120 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K E K N N S L D V A I V Y R K E E A L A I V S D D A K Q L . . D K S Y R K C Y D F N D Q . . . . K E G E N K T BTA115 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L D K D K N N S I D I L I K V A A I V S D D A K Q L . . K K D F N F E Q A C K L I E K K A . . . . D E N G E R R N BTA112 cc c c KddddddG KddG B KG KG KG KG KdG B KG KdddG KG B T Y F L N S L D I M I V E E I I L T E E K Q L C K T L T E Y A L G A K R E K V N L D F K V R . . . . E A D A G K V A K T

BTA129 cc c c JIIIIIIH JIIH C JH JH JH JH JIH C JH JIIIH JH C T Y F L N N L D V M I Y D A L A I V D R L Y R T D I E N L E G N K F A L V L E E Q S N Q A W L E A E D S D A D F E Q R V

iiiiiiiiiiiiiiiiii iii iiiiiiiiiiiiiiiiiiiii

. fBTA121 3 9 0 4 0 0 4 1 0 4 2 0 4 3 0 4 4 0 X X X X X X fBTA121 c c c A A DF A DF DF A DF A A A DF A A Y F Y R N E S I R L Q I N F L K A A L R F K G Y N G T K . G F T D G H F M T D G D K R Y I I F D Q A R V A K A R G A T G BTA121 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R L Q I N F L K A A L R F K G Y N G T K . G F T D G H F M T D G D K R Y I I F D Q A R V A K A R G A T G BTA121-His c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R L Q I N F L K A A L R F K G Y N G T K . G F T D G H F M T D G D K R Y I I F D Q A R V A K A R G A T G BTA116 c c c B B KG B KG KG B KG B B B KG B B Y F Y R E S I R A L Q L F I K A V L E R L Q G N H D N . K S E G E S F T N D S N K R R G D V S N K A K A S G A A G G G S BTA123 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R A L Q I N F R A A F T L Q G D H D N . K A S G E Y A I G N G D K R D I G S N N R A K F D E E A A A A A BTA126 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R A L Q I N F I A A F T L Q G N H S N . Q S S V D Y F T G N G D K R D N A E K A K A E A A Q G A A A A V BTA127 c c c B B KG B KG KG B KG B B B KG B B Y F Y N E S I R N F L K A A I F R L K D N H D T N Q . S S D K F Y A F . F I K I I K S G S I E D K K V L R A A A A A K A BTA119 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R A L Q L S F I R A A F T L Q G Y S D N . K S A D D Y A S Y Y P G K S G P K D D K R A A A A K E A A A . BTA118 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R A L Q L S F I R A A F T L Q G Y S D N . K S A D D Y A N H Y P D E S G P K D D K R A A A A K E A E A . BTA117 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R A L Q L S F I R A A F T L Q G Y S D N N K S A D G Y A G R D L Y E S G P K D D R R A K A A K A A E A . BTA122 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R A L Q L S F I K A A F M L K D Y H D N . R S S D D Y F N H Y S Y E S G F K E D K K A A A A E A A K . . BTA124 c c c B B KG B KG KG B KG B B B KG B B Y F Y R E I R A L Q I I K V L K R V K D N A D D D . K P L V E V R L N N R D V L G R L N G A T A V G K S E E E A . . . . BTA120 c c c B B KG B KG KG B KG B B B KG B B Y F Y R N E S I R A L Q L S F I K A A F T L Q G Y H D N . K S A D D Y S N R Y P D E S G S E E D K K A A A A A A A E R A BTA115 c c c B B KG B KG KG B KG B B B KG B B Y F Y N S I R A L I F I R N F E L K G M Y H D A . L S I D E C Y A E H Y H N N Y A D Y G V E T . . . . BTA112 c c c B B KG B KG KG B KG B B B KG B B Y F Y R E L R A S N Y L K I L A S Y N N A F I G N . A S G A D N I Y R F M K I . N I A I N K K R D P . R V L D V Y T W L BTA129 c c c C C JH C JH JH C JH C C C JH C C Y F Y K K L L Q F I K A I I D D E R D N K L H F E N . E F L S E V Y K F M K N I C Y D K V L K T Q H I A F K K L Y K K L α5 η1 α6 α7 η2 α8 α9 η3 η4 α10 α11 α12 α13

fBTA121 fBTA121 S G . S . . . . BTA121 S G . S . . . . BTA121-His S G . S . . . . BTA116 . . . . BTA123 K E . A K E A E E A A A A . . . A . . E R A K E A K E A E A A K A A E A A K R A K E A K E A E A A A A A K R BTA126 K S . S G A . . . . BTA127 S G . A A G . . . G S . . . . BTA119 . . . . BTA118 . . . . BTA117 . . . . K . . . . BTA122 . . . . BTA124 . . . . BTA120 K E . A K E . . . A A V A A E . . . BTA115 . . . . BTA112 S D E D K G V I D K I Q G I V T D S N I G H A K G Y K T Y T D S E F N D L L N N L G A S K T R S M I T . . . A F L R BTA129 S S V Q K R G L D Y I R I V V T N F N I G V L . . H K T Y N A I E F E S L L G S L G A T R V E R I I D . . . F H L N fBTA121 fBTA121 . . . . BTA121 . . . . BTA121-His . . . . BTA116 . . . . BTA123 A K E A K E A E A A A A A K V E E S T E A G E P R E G S G T D E E S G A T G S G S . . . . BTA126 . . . A G G G S . . . . BTA127 . . . S . . . . BTA119 . . . A K A A K A E E G S G T D E E S G A A D G G S . . . . BTA118 . . . A K A A E A E E G S G T D E E S G A A D G G S . . . . BTA117 . . . E A A V A A E A G E P R E G S G T D E E S G A A D G G S . . . . BTA122 . . . A E E G T K T D E E S G T A D G G S . . . . BTA124 . . . D K A A A A A V K A E G S T E A G E P G E G S G T V E E S G A T G G G S . . . . BTA120 . . . A E E S T E A G E A E E S T E A G E P R E G S G T D E E S G A A D G G S . . . . BTA115 . . . . BTA112 A N V L R E M . . . . A L E A I N T V P A G T A K Q N F Q . . N R F N A L S N G Y A L R L K G L F N S L N H D M I Y D S BTA129 V L K A K K D . . . . A Q S A I S S L P E S M T R H N L Q . . R L F D A H S N G Y E L H L K E L F H I N F P Y Q V Y Y S fBTA121 fBTA121 . . . BTA121 . . . BTA121-His . . . BTA116 . . . BTA123 . . . BTA126 . . . BTA127 . . . BTA119 . . . BTA118 . . . BTA117 . . . BTA122 . . . BTA124 . . . BTA120 . . . BTA115 . . . BTA112 V V N D E Y S E K F L R I K D D I R H L S S N T A D S T A S S N BTA129 V A R S S Y V D L Y F E I I N Y V G K I Q . . .