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