Scientific Reports

Supplementary Information

Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori

Rintaro Suzuki1, Zui Fujimoto1, Takahiro Shiotsuki2, Wataru Tsuchiya1, Mitsuru Momma1

Akira Tase

,

1, Mitsuhiro Miyazawa3 & Toshimasa Yamazaki

1*

1Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai,

Tsukuba, Ibaraki 305-8602, Japan

2Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi,

Tsukuba, Ibaraki 305-8634, Japan

3

Insect Mimetics Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba,

Ibaraki 305-8634, Japan

*Correspondence and requests for materials should be addressed to T.Y. (tyamazak@nias.affrc.go.jp).

This Supplementary Information contains five figures and four tables.

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C C G C C G C C G C C G

G G G G

G G G G

C C G C C G C C G C C G C C G C C G C C G C C G C C G C C G

G G G G G G G G G G

G G G G G G G G G G

D A P D M LS ATE FLEKT IP YDI IDPLVV SLDVIA D A P D I LS ATQ FLEK VPEYDIR IDPLIIPSLDVAA D E V P D I LS ATE FLEKT P EIK IDPLVIP L VVV E L P D I IS ATQ FLD T IPEY IK LDPI IPSLE I E

GI I FKN I LK I D D K LL L I G I GL FKN V LK I D R D RK LL L V I GL FKN I LK I D K D KK VL L I I L V Y N V K I K A G I

A V V G H V E L L I A I A G H V E L L D A A A Y V EI I L I A A A G H A EI V L D

I D AQ AVP V IPDLGI LDPL I I L I V I M I EL I LDP D L V I D A A V PE GL LDPL VE M V V L D I AL I IPEL I VDPI L V V V D L LV L APEMGI AEPI ID L I L I N I IE LP IPEL V DPV LD I I V D M Q AMP LPEIGV LD M LD V L I D L AQ AVP L I DLGI MDPM V V V D L AQ MVP L LPDLG LDPL VE I V L V D M Q ALP LPD GV MD L LD L

GL L FKD V L V VK D KK AV V L G Y L V V M KN V LK V DAR V I I G F A G V FKD L L NLK D KK AA L V Y L V V I A VK LK D K A I I G Y V L G A FKD A A I NVR D H L I A Y L GL L F M LK LK D H L M L Y A I GL D L K I DVK D KR V I G Y A V GL M FRD V LR V NLR LL V L G Y L I GL L D I MK I L D KK V V L G Y A I GL K L LK I NVK D KK V A V G Y A I

I I I G H I EV L N M L Y I DV L I L I G H I EV L L N V L L M G Y D A L V V G Y L V V L N I V I G H L I L L N L L M G H L DV L L N I I I I G Y V E L N I L L M G L DV M L N L L I L G H DV L L N

D A P M S AT F EKT YD V S D I D A P I S AT F EK YD R I PS D A D E V P I S AT F EKT E K V P V E L P I S AT F D T Y K PS E E

A V R LN T NQ S F M T TV KTKAD V DIV ELT Q I I H LN T NQ S F M T SV KT AD V DVV ELSKQ M V IN NQ S F M T SV KTKA V DL E TKQ I R L T NQ S F L V KTK N LV EL K

SKSF IG Y Y L DD VQ FE ET TC IGEP I D AL SKSF S IG Y Y L D V FE G ET SC IGEP V E AL K F T LG Y Y D E F G E C IGEP V E AL SKTY S G Y Y V DD VE YE G ET SC GEP L AL

FI A A P V LG HL V M L D YI A G E L I A I L F L L MG D YI I P L D V A P IG D FI A A P L LG V A M P L LG E V A P G D

T R V T Y G LLV K A AD F G A K L MD Y G ILV T K L LE Y L R T V LE S Y G LLL K T H L D S Y G LLI K T K V E S Y G ILV H R L D T Y G LLI K L L Y G LLI T K V L T Y G ILI

LPI G K RD I D K H W Y A F N F GN L V D K VR F M N IPI K KN L D K K H Y A F N D V G I K D MK Y F N F G V A G A D R LK F I N N L I G N V K K LK Y F N F GN LPI G K KN I K D K I Y F A F N F GN LPI G R RD M E H W V Y N F GN LPI G K KN I K E K I Y Y A F N F GN LPI G K KN I K E V Y F A F N F GN

gs GD LLK KLG ... Q S Q S.K Q WP T P.S QG LFE STQ ... A R Q AC.R P Y. GG FFN YKS ... K N T C.N Y NT A E K D. SKLN STE DVS ... E K V N Y.Q N K T KS ..

D ..Q Q K...A H E .......D F ..K T... Q N A .......S FD V ..Q D... I N A K F .......K N N N K F ..K H T VRD...T A NF V FT E K P S.......

.. T LYT DT.N A R G N KN N QP F S.. K T SS SS

.. A VMN Q . G K S D QD SK K Q I S.. A N NP A

..N V N PYI K . SSQ G SFTKK D.K V S ENA .. D E GE QK

.. T EVT E . E G A S S KT K P V ..F T S SST E

ASAP TP KVG ...SA FVAS A I AA. KP H SV NGNQ... AIEISK KV NRNSLDLND VEA RDG EK AT. E D PP FFQ E K EYKNN SVVGLGLPS SS SRK PNLND LKS RNS HQFSL. D R PP Y N V Y P.N AFVDT GK SLK ...EE TKSL NN NE SST D PP A KN S T ... NI GGK SRQ PNVDA LRSFNN DY KG. EE V E S A GAG DDAYLPP EA EFS ENLEQ KEQ KS EFTK. D PST H D N DG...MHCIESVFSVLP DK KLE ...SA ITAF N VFFS. EV V E Y EFD ... TDLAK TP RPK ...TA KSS K F AA. A ET T GR NTVQ... LPD QK NLT ...AV KTS L Q TD. SKV Y A K D ... GVLP EK NLE ...SA TSAF Q TFVA. H EV V D DFAFD ...

.G E TI SGC..H EG N PA... KQ TIKCS T D K S K ......QIA LI IY E GS..T H VR EDNFYME DLSAPS V R D KGE QYN ......RN F V NYFT SSL..T Q F ... II QALVN D NNT D S R ......LGL D LLEGQ TC..KFSS I ... EIG YEFTCD D E K K FSESP IKNLLGPD Y IH S SNM..T T S ..T QFQ TLYGPH RAR RSS V .......N TKAL GSHLKEFK F G ... GNFK AFISN TAE E D K .......S F LS GR H NA..T D W T ... RFD IYHLDS K H T G S .......A D TT NC..V L RLH .....KT DLKCS V E T G Q .......A ALT AE T DT..I DK SV A... QIQ ICHVS Y K K K S K .......S QF L EGK GA..V D W K... NIE DFHLD K H T G R ......

. E E KYN V KTANDLVTVTGA P W ES KHT ..KTG H QF.E

. K KAY DFNTS S IFTWKLDGVPE N.GTDT KS YMRP ...GN ISH .N ENPET

. KSS DSF TL N NFYYEDIEDA V W FN DVE ...EK V R .E L . GTT H E NGK Q EK Q S KFPVYAQ RD EI CDYSKIK YKILGKTK Y .D L E. R S G EYWGEYHG K K YFRGAPYER. T QQLKLD S ...KD KMG .E D S . Q K K DAL NCV D E ESKLNQV DNN D GTPSYK K ...EKTT D .K . T D QMK Q HFYIEYDVE AE D V KK DFD ..REN H E .S . E T KYR I K ILDVEERIV. DR W SD KHSA D..VSK E QF.Q . T D DTT R Q IYPFNLV NS DV D SS RYS ..KDN H H .T . T D QMK H H VVSYEME DA VD IFKK TVTF ..KDN Q G .T

NMR

BmJHBPMsJHBPHvJHBPGmJHBP

NMR

BmJHBPMsJHBPHvJHBPGmJHBP

NMR

BmJHBPMsJHBPHvJHBPGmJHBP

X-ray B

2RCK A

X-ray B

2RCK A

X-ray B

2RCK A

X-ray A

2RCK B

X-ray A

2RCK B

X-ray A

2RCK B

α1 β1

β2a 310 310β2b β3

β4 β5a β5b

β5b

β5c

β5c

α2

α1SS β0a 310 β0b β1

β2a 310 β2b β3

β4 β5a α2

3E8T

3E8T

3E8T

3E8W

3E8W

3E8W

α1 310 β1a β1b

β2a 310 β2b β3

β4a β4b β5 310

an-0128an-0147an-0895an-0921brP-1649brP-2095ce-0330e96h-0303wdS3-0639EpTo1

an-0128an-0147an-0895an-0921brP-1649brP-2095ce-0330e96h-0303wdS3-0639EpTo1

an-0128an-0147an-0895an-0921brP-1649brP-2095ce-0330e96h-0303wdS3-0639EpTo1

-2111

55555555

109109109109

111111111

536163545759605451

107119118115113115114106105106

1

52

.. ..

D L IA V I LP D I IV V I LP D N L IV V I P Q IV I LP

N N V IV V V VP N N L A A VP N I I V L V N N V VP N L L M L IP N N L AI V I V N N V IM I N N I MV I VP N N L LL V V P N N V VM I I VP

D L E L R ICK EA YI VHN RA AKI S FF NL D D YE A R SLCK EL YV VHN RAVAKI TAFF DV D D M YE R TLCH EA YV IHN RAVAKL AFF DI D D I Y L R TACR E YA VHN RAAARI SAYF NV

K L V FV WK M EV PP IV Y AEEL REL FL Q WR EL P K F D I K L I L Q V EV P LM DEL EA V FL E WK M AM K F A L L FI E L EM P LA H DEL KEL FA E W Q M DL PP IV K F I KEL I FL E WKQ V E P A F LEDI RDL I FA WRE EV PP IV K F I DL K L M FM Q WK E P M LEDI KEL M FL Q WKQ E P AA H I EI

EK SGNNS EP M .P KT QAA C S S A S E N LK P TTHYRK AH VG QR C D S T N LN A AKA KP A KVA KK A T F KE L KL S FKK FTE GK TEG KQ TV VS K K

V ATP EE S.. D Q A RS SE VAA DAL KA YLQ TNLGTS .. V YR L Y QSNWDKIGTNV N I SK Y Q FPSGT N GEQ NKI G..LSQ D G T CGS KA VEN GILLEQ SF MPE Q SKL TT .. F DTFGRSFFDK DIFYSYTS F GT KHYLTDDFTSIAKP AV QAA NL ST.. AQ K K E KRD DK SRY D ILQH Y VVD ADTTLQ .. Q D A KQ KT VKA N F AS TTQQ IKGYSP SKI HS .. T FGR DAT KKFFKN NIF EKNS AIL AKT HD S.. FQ A KA SK IHETF L DK K ALE Q GDV LT .. A TQ FGK EIP EM YNT KTYLKSQ ANL SDT LT .. SE FGK EA KK FKN K FLAK A ANV

SS

NMR

BmJHBPMsJHBPHvJHBPGmJHBP

X-ray B

2RCK A

X-ray A

2RCK B

α3a α3b 310

α3a α3b

3E8T3E8W

α3a α3b 310

an-0128an-0147an-0895an-0921brP-1649brP-2095ce-0330e96h-0303wdS3-0639EpTo1

170170169170

169179178180173176176167167168

Supplementary Figure S1 | Sequence alignment of JHBP/Takeout family. BmJHBP, MsJHBP,

HvJHBP, and GmJHBP denote JHBP of Bombyx mori, Manduca sexta, Heliothis virescens, and

Galleria mellonella, respectively. EpTo1 denotes Epiphyas postvittana Takeout protein. Other nine

sequences are deduced for Bombyx mori Takeout proteins reported previously (13). Secondary

structure elements of BmJHBP complexed with JH II in the crystalline state (X-ray) and with JH III

in solution (NMR) are shown above its sequence while those of GmJHBP (15) and EpTo1 (14) in

the crystalline states are shown below their sequences. Residues colored red assume cis

configuration in their three-dimensional structures. Highlighted in black backgrounds are residues

strictly conserved and semiconserved for both the JHBP and Takeout subfamilies. Residues

highlighted in purple and green backgrounds are conserved for the JHBP and the Takeout

subfamily, respectively. The BmJHBP residues essential for JH binding are indicated by asterisks

(*) above its sequence. Alignment of multiple sequences and structures was initially achieved by

the program MAFFTash (http://sysimm.ifrec.osaka-u.ac.jp/MAFFTash.3/) and modified manually

so that the secondary structures are well-aligned and most gaps are located within loops.

a

b

c

Supplementary Figure S2 | Comparison of (a) the crystal structure of the gate-closed

conformation of the Bonbyx mori JHBP in complex with JH II with reported crystal

structures of (b) Epiphyas postvittana Takeout protein (EpTo1) (PDB ID: 3E8T) and (c)

human cholesteryl ester transfer protein (CETP) (PDB ID: 2OBD). JHBP and EpTo1 are

displayed with the same orientation after three-dimensional superposition. CETP is displayed so as

to show maximum similarity of its N-terminal domain with JHBP. Bound ligands are shown in stick

models. Internal hydrophobic cavities of JHBP and the N-terminal domain of CETP are divided into

two smaller pockets at the middle by hydrogen-bonded side chains and by hydrophobic side chains,

respectively. These residues are shown as space filling structures. In the EpTo1 structure,

Takeout-specific region composed of motif 1 (residues 96-113) and motif 2 (160-182) is

highlighted.

15N

(ppm

)

10 9 8 7

130

125

120

115

110

105

1H (ppm)10 9 8 7

1H (ppm)

a b

c

1501005010

200

0.2

0.4

Residue Number

Δδ

(ppm

)

NMRα3β4 β5a β5bβ5c α2β2a β2b β3α1 β1aβ1b

Supplementary Figure S3 | 1H-15N HSQC spectra of (a) JH III-bound and (b) apo-JHBP in 45 mM

sodium phosphate buffer, pH 6, collected at 35 °C on a Bruker DMX 750 spectrometer. Signals

shown in red are folded under the experimental conditions employed. (c) Changes in backbone 1H

and 15N chemical shifts of JHBP upon complex formation with JH III. Weighted chemical shift

differences between the apo- and JH III-bound JHBP, calculated with a function ∆δ = [∆δHN2 +

(∆δ15N/5)2]1/2, are shown against residue number. No NMR signals were observed for the shaded

regions of the protein in the apo-form. The secondary structure elements of the JH III-bound form

in solution are presented at the top of the figure.

a b

c

3

4

57

Supplementary Figure S4 | NMR solution structures of B. mori JHBP-JH III complex. (a)

Superimposed backbone (N, Cα and C’) atoms of the final 20 NMR-derived structures with the

lowest energies for the JHBP-JH III complex. The backbone coordinates were superimposed against

the representative structure, which showed minimum rmsd with the mean structure, using residues

1-225. JH III atoms are shown in pink. (b) A ribbon representation of the representative structure of

the complex. Side chains of the residues forming the bottom wall of the JH-binding pocket are

shown as ball-and-stick models and disulfide bonds as stick models. The bound-JH III molecule

(pink) is depicted as a space-filling structure. (c) Superposition of the JHBP-bound JH III in

solution. The observed L-shaped structures and crankshaft-like motions of the bound JH III in

solution are similar to those for the bound JH II in the crystalline state (Figure 2C). Note that these

L-shaped structures are accomplished by gauche(-) conformations about the C3-C4 and C4-C5

bonds instead of the successive gauche(+) conformations observed for the JHBP-bound JH II in the

crystalline state. For the isolated JH III, conformational energies of the L-shaped structures with

gauche(+) conformations and with gauche(-) conformations are comparable as calculated using the

CHARMm force field (Supplementary Table S4). Nearly isoenergetic L-shaped structures in rapid

equilibrium on the NMR time scale would make a favorable entropic contribution to complex

formation. These findings suggest that JH has a certain motional freedom even when bound to

JHBP.

a

1501005010

200

0.2

0.4

Residue Number

Δδ

(ppm

)NMR

α3β4 β5a β5bβ5c α2β2a β2b β3α1 β1aβ1b

b

Supplementary Figure S5 | (a) Comparison of backbone amide 1H and 15N chemical shifts of the

JH III-bound B. mori JHBP between pH 6 and pH 4. Weighted chemical shift differences between

pH 6 and pH 4, calculated with a function ∆δ = [∆δHN2 + (∆δ15N/5)2]1/2, are shown against residue

number. Red bars represent Asp and Glu residues. The secondary structure elements of the JH

III-bound form in solution are presented at the top of the figure. (b) Mapping of the perturbed

residues of the JH III-bound JHBP upon changing pH from 6 to 4. The perturbed residues shown in

red, magenta, and pink are classified according to the magnitude of the perturbation, ∆δ > 0.1, 0.08

< ∆δ < 0.1, and 0.07 < ∆δ < 0.08 ppm, respectively. It is noteworthy that most of the perturbed

residues are solvent-exposed Asp and Glu shown as ball-and-stick models, and their surrounding

residues. Proline and N-terminal residues are shown in light-green. The bound JH III molecule is

shown as a stick model.

Supplementary Table S1 | Summary of Data Collection and Refinement Statistics of the Crystal Structures of the JH II-bound and apo-JHBP

Se-Met JH II Apo

Data collection

Space group P21212 C2 1 P63 22

Unit cell parameters

a (Å) 54.8 164.0 90.9

b (Å) 114.8 46.8 90.9

c (Å) 194.1 72.2 138.3

β (°) 102.5

Beam Line PF 17A PF-AR NE-3 PF-AR NE-3

Wavelength (Å) 0.97888 1.00000 1.00000

Resolution (Å) 50 - 2.80 ( 2.93 - 2.80 )

50 - 2.20 ( 2.28 - 2.20 )

50 - 2.20 ( 2.28 - 2.20 )

Total reflections 406072 173801 366645

Unique reflections 30555 (2720) 24811 (1826) 17925 (1797)

R-merge 0.115 (0.499) 0.075 (0.312) 0.059 (0.336)

Completeness (%) 98.4 (88.9) 90.7 (68.1) 99.8 (100.0)

Average I/σ(I) 24.5 (2.5) 20.2 (5.2) 57.3 (11.3)

Average redundancy 13.3 (8.7) 7.0 (6.1) 20.5 (10.9)

Structure refinement

Resolution (Å) 80.1 - 2.20 ( 2.26 - 2.20 )

78.7 - 2.20 ( 2.25 - 2.20 )

R-factor 0.239 (0.236) 0.234 (0.221)

Rfree-factor a 0.291 (0.281) 0.254 (0.263)

RMSD from ideal

Bond lengths (Å) 0.015 0.017

Bond angles (°) 1.63 1.58

Ramachandran plot (%)

Favored regions 95.4 96.5

Allowed regions 3.9 2.5

Outlier regions 0.7 1.0 a R free

-factors were calculated using 5% of the unique reflections.

Supplementary Table S2 | Comparison of (φ, ψ) angles of the hinge point residues for the movement of the gate α1 helix of B. mori JHBP between the apo- and JH II-bound forms in the crystalline state

(φ, ψ) angles

Residue apo-form JH II-bound form a

Thr28 (-71.4°, -31.5°) (-82.9° ± 6.6°, -9.8° ± 1.5°)

Ser29 (-94.0°, -23.7°) (-50.0° ± 2.1°, -22.7° ± 12.7°)

Lys30 (-112.0°, -18.3°) (-120.7° ± 16.5°, 16.7° ± 2.6°)

Gly31 (81.9°, -173.0°) (97.1° ± 12.9°, 158.0° ± 2.2°) a

Average angles of the two structures in the asymmetric crystal unit.

Supplementary Table S3 | Statistics of the NMR Solution Structure of the JHBP-JH III Complex

NOE restraints Unique Ambiguous Total a

Intraresidue 721 1127(527) 1848

Sequential (|i – j| = 1) 805 1603(750) 2408

Medium range (1 < |i – j| < 5) 733 1606(732) 2339

Long range (|i – j| ≥ 5) 1654 3493(1564) 5147

Total 4274 7468(3212) 11742

Hydrogen bonds 89

Dihedral angle restraints

φ Angles 147

ψ Angles 147

RMSDb from mean structure Residues 1–225

Backbone atoms 0.32 ± 0.04 Å

All heavy atoms 0.64 ± 0.05 Å

Ramachandran plot All residues

Favored regions 90.3%

Allowed regions 7.8%

Outlier regions 1.8% a Numbers of amiguous NOEs. Numbers of ambiguously assigned peaks are indicated in parentheses.

b

Root mean square deviation of superimposed atoms, calculated by CYANA.

Supplementary Table S4 | CHARMm Energies Estimated for L-Shaped Conformations of Isolated JH III

NMR 1 NMR 2 X-ray A Xray B Extend

C3-C4 -52.4 ± 7.8 (-54.2 ± 6.2)

-81.4 ± 12.2 (-77.1 ± 11.2) 57.5 (58.0) 50.5 (52.9) -118.8

C4-C5 -56.6 ± 3.2 (-53.2 ± 4.7)

-70.5 ± 0.7 (-48.0 ± 6.0) 60.9 (59.1) 57.0 (76.0) 177.3

C7-C8 113.9 ± 1.0 (119.4 ± 5.0)

112.7 ± 0.7 (-114.7 ± 14.0) -6.5 (-0.3) -113.9 (-89.5) -115.0

C9-C10 -82.4 ± 2.5 (-60.0 ± 4.9)

176.5 ± 0.5 (137.5 ± 21.9) 171.3 (157.8) -82.1 (-127.0) 141.7

Energy (kcal/mol) 46.3 ± 2.2 45.3 ± 3.3 50.6 47.7 41.2

Coordinates of the JH III molecules in 20 NMR models were used as initial structures for energy minimization calculations

of JH III with gauch(-) C3-C4 and C4-C5 bonds. The resulted JH III structures are classified into two groups by the torsion

angles about the C7-C8 and C9-C10 bonds (1 for 15 structures and 2 for five structures). Two JH II molecules bound to

JHBP A and B chains in an asymmetric unit of X-ray structure were energy-minimized and ethyl group bound to epoxy

carbon was substituted by methyl group to prepare initial structure of JH III with gauch(+) C3-C4 and C4-C5 bonds. An

initial structure of extended JH III model was build to have 180° torsion angles for all rotatable bonds. Torsion angles of

initial structures are shown in parentheses. The energy minimization calculations were executed with CHARMm forcefield

and partial charges estimated by use of MMFF94.