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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
Muscle and the structure of the sarcomere
MyosinAktinTitin
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
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
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
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.)
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
Weak interaction in the interface
A168 A169
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
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.
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)
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
Structure-based sequence alignment of PB1 domains
PB1 domain of NBR1
OPCA-motif
D50 E52
E54
100um
red: negative charge
P6322
E63
1.55 Å
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
Structure of the NBR1/p62 PB1 complex
NBR1 p62
PEG/Na Formate/NaAcetate PEG/Na Formate/NaAcetate+ CdCl2
OPCA motif mediating interaction
NBR1 p62
E54
D50
E52
Y9
K7
R96
Interaction additionally supported by E63 (NBR1) and R21 (p62)
Electrostatic surface potential of the PB1 domains of NBR1 and P62
red:- charge
blue:+ charge
Summary
PB1 ZZ UBACC CC
PB1ZZUBA
RING MFC
BboxAR
M-line Titin
A169A168A170 TK
M1NC
PB1 ZZ UBACC CCNBR1 dimerMURF-1
p62
Acknowledgements
EMBL, Hamburg Matthias WilmannsInari KursulaSantosh PanjikarKatja Schirwitz
King’s College, London Mathias GautelStephan Lange
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