structural studies on domains in proximity to titin kinase and
TRANSCRIPT
![Page 1: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/1.jpg)
Structural studies on domains in proximity to titin kinase and domains involved in its
downstream signaling pathway
Simone Müller, Vienna, June 28th 2006
EMBL Hamburg
![Page 2: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/2.jpg)
Muscle and the structure of the sarcomere
MyosinAktinTitin
![Page 3: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/3.jpg)
The muscle sarcomere- and the giant muscle protein titin
adapted from Labeit & Kolmerer (1995)
Titin: ~300 domainsup to 38138 aa and 3.7MDa 1.2 µm lengthZ-disk titin
M-line titin
![Page 4: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/4.jpg)
Signaling pathway of Titin kinase (TK)
PB1 ZZ UBACC CC
PB1ZZUBA
RING MFC
BboxAR
M-line Titin
A169A168A170 TK
M1NC
PB1 ZZ UBACC CCNBR1 dimerMURF-1
p62
Titin kinase in a semi-open stateAssociation of the NBR1 dimers and the regulatory α-helix R1 of TKInteraction of NBR1 and p62Interaction of MURF with p62 and A168-A169Phosphorylation of NBR1 and p62 (and MURF?)
Lange et al. (2005) Science
![Page 5: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/5.jpg)
Titin Ig domain M1I41
A169A168 A170 TKM1N C
1.7 Å
A
A’
B E DC F G
C’
AA’
GF
CB
E
D
C
N
![Page 6: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/6.jpg)
Tandem Ig domains A168-A169
A169A168 A170 TKM1N C
RING finger BBox C C C C AR MuRF-1
MuRF: Muscle-specific RING finger proteinsMuRF-1 interacts with Titin A168-A169; potential regulator of TK (Centner et al. (2001) J. Mol Biol.)
Interaction with diverse sarcomere proteins (Witt et al. (2005) J Mol Biol)
Localisation: cytosol and nucleus (atrophic cond.) (Pizon et al. (2002) J Cell Sci.)
![Page 7: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/7.jpg)
Tandem Ig domains A168-A169
A168 A169
• continuous β- strand (blue)• bulge between strand A and A’ in A169 (yellow)
I2221.99 Å
C2222.5 Å
A168 A169
![Page 8: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/8.jpg)
Weak interaction in the interface
A168 A169
![Page 9: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/9.jpg)
Structure-based sequence alignment of titin Ig domains
A A' B C C‘ DA169 ----PAKIHLPKTLEGMGAVHALRGE-VVSIKIPFSG-KPD-PVITWQKGQDLIDNN--G-HYQVIVTR-A168 ---MAPHFKEEL-----RNLNVRYQS-NATLVCKVTG-HPK-PIVKWYRQGKEIIAD--GLKYRIQEFKG Z1 MTTQAPTFTQPL-----QSVVVLEGS-TATFEAHISG-FPV-PEVSWFRDGQVISTSTLP-GVQISFSD-Z2 --TAPPNFVQRL-----QSMTVRQGS-QVRLQVRVTG-IPN-PVVKFYRDGAEIQSS--L-DFQISQEG-I1 -SMEAPKIFERI-----QSQTVGQGS-DAHFRVRVVG-KPD-PECEWYKNGVKIERS--D-RIYWYWPEDI27 -AMALIEVEKPL-----YGVEVFVGE-TAHFEIELSE-P-D-VHGQWKLKGQPLAAS--P-DCEIIEDG-M1 ---GAMVS-GQI-----MHAVGEEGG-HVKYVCKIENYD-QSTQVTWYFGVRQLENS--E-KYEITYED-M5 ------RILTKP-----RSMTV-YEGESARFSCDTDG-EPV-PTVTWLRKGQVLSTS--A-RHQVTTTK-
E 310 F GA169 SFTSLVFPNGVERKDAGFYVVCAKNRFGIDQKTVELDVAD--- 99 A168 GYHQLII-ASVTDDDATVYQVRATNQGGSVSGTASLEVEV--- 96 Z1 GRAKLTI-PAVTKANSGRYSLKATNGSGQATSTAELLVKAE-- 100 Z2 DLYSLLI-AEAYPEDSGTYSVNATNSVERATSTAELLVQ---- 94 I1 NVCELVI-RDVTGEDSASIMVKAINIAGETSSHAFLLVQAK-- 98 I27 KKHILIL-HNCQLGMTGEVSFQAA----NTKSAANLKVKEL-- 92 M1 GVAILYV-KDITKLDDGTYRCKVVNDYGEDSSYAELFVKGVRE 97 M5 YKSTFEI-SSVQASDEGNYSVVVENSEGKQEAEFTLTIQK--- 91
![Page 10: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/10.jpg)
Involvement of the bulge in A169 in binding to MuRF-1 ?
K104E and E107R ?
Pull-down assays
experiments: Stephan Lange, King’s College London
A168-A169 E107R mutation reduces the interaction with MuRF-1.
In vivo colocalization studies and split-GFP experiments confirm the pull-down assays.
![Page 11: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/11.jpg)
Interaction of NBR1 and p62
OPR = octicosapeptide repeat, 28 aaPC = Phox and CdcAID = atypical protein kinase C (PKC)- interaction domain
OPCA = OPR/PC/AID motif is located within the PB1 domain
OPCAmotif
PB1 ZZ UBACC CC
PB1ZZUBA
p62 NBR1
Interaction has been shown by biochemical and cell-biological experiments:Lamark et al., JCB (2003), Lange et al., Science (2005)
![Page 12: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/12.jpg)
PB1 PB1K OPCA
NBR1p62 DDAA
OPCAPB1 KOPCAXX
PB1OPCAPB1 K
type-Atype-B
D
Basic back
DK
PB1 KOPCAPB1 KOPCA PB1 KOPCA
type-AB
Acidic hairpin
Cdc24p p40phoxaPKCNBR1
Bem1pp67phoxpar6
p62
front-to-back interactions
p62C
B
A
Model of the PB1 interaction
Ito et al. (2001) EMBO J.Wilson et al. (2003) Mol. Cell
![Page 13: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/13.jpg)
Structure-based sequence alignment of PB1 domains
![Page 14: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/14.jpg)
PB1 domain of NBR1
OPCA-motif
D50 E52
E54
100um
red: negative charge
P6322
E63
1.55 Å
![Page 15: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/15.jpg)
Complex formation of the PB1 domains GF Superdex75 HR 10 30118:1_UV1_280nm GF Superdex75 HR 10 30115:1_UV1_280nm GF Superdex75 HR 10 30118:1_Fractions GF Superdex75 HR 10 30118:1_Inject GF Superdex75 HR 10 30116:1_UV1_280nm GF Superdex75 HR 10 30118:1_Logbook
0
20
40
60
80
100
mAU
0.0 5.0 10.0 15.0 20.0 25.0 mlD1 D2 D3 D4 D5 D6 D7 D8 Waste
Superdex 75 10/30
25 mM HEPES pH 7.5150mM NaCl
5 mM DTT
Blue: complexRed: p62 DDAA PB1Green: NBR1 PB1
ITC: Isothermal Titration Calorimetrycell: 0.05 µM p62DDAAsyringe: 0.50 µM NBR1Kd= 12 nM ± 1 nM
![Page 16: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/16.jpg)
Structure of the NBR1/p62 PB1 complex
NBR1 p62
PEG/Na Formate/NaAcetate PEG/Na Formate/NaAcetate+ CdCl2
![Page 17: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/17.jpg)
OPCA motif mediating interaction
NBR1 p62
E54
D50
E52
Y9
K7
R96
Interaction additionally supported by E63 (NBR1) and R21 (p62)
![Page 18: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/18.jpg)
Electrostatic surface potential of the PB1 domains of NBR1 and P62
red:- charge
blue:+ charge
![Page 19: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/19.jpg)
Summary
PB1 ZZ UBACC CC
PB1ZZUBA
RING MFC
BboxAR
M-line Titin
A169A168A170 TK
M1NC
PB1 ZZ UBACC CCNBR1 dimerMURF-1
p62
![Page 20: Structural studies on domains in proximity to titin kinase and](https://reader034.vdocuments.net/reader034/viewer/2022051319/589af8c41a28ab91468bda54/html5/thumbnails/20.jpg)
Acknowledgements
EMBL, Hamburg Matthias WilmannsInari KursulaSantosh PanjikarKatja Schirwitz
King’s College, London Mathias GautelStephan Lange