scientific reports supplementary information · ni ggk srq pnvda lrsfnn dy kg. ee v e s a ga...
TRANSCRIPT
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. ([email protected]).
This Supplementary Information contains five figures and four tables.
.. . ..
... . .. ... . ..
... . ... ... . ...
.. ... ....... .. ... .......
. .. ... ......
. .. ... ......
... . . ... . . ... . .
.. ... ....... .. ... ....... .. ... .......
.. . ..
.. . ..
.. . ..
.. . ..
. .. .
. .. .
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.