binding to negatively curved
membranes
Cell biology with bacteria?
5 µm
Localization of cell division proteins
Rut Carballido-López
GFP-MinD
How do proteins localize to cell poles ?
(DivIVA as model system)
DivIVA-GFP
(lack of) Information from secondary structure prediction
164 amino acids, mostly helical
secondary structure prediction by PSIPRED
coiled coil prediction by LUPAS
multimerization via coiled coil regions
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes
Binding to another (membrane) protein?
DG = DivIVA-GFP
V = membrane vesicles
20 %
membrane vesicles
Lip = liposomes
30 %
D = DivIVA
70 %
G = GFP
Biacore (surface plasmon resonance) with L1-chip
T = min
amphipathic helix of N-terminus (60 aa)
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes
Edwards, 2000, EMBO
Cardiolipin Domains in Bacillus subtilis
Kawai, 2003, J. Bac.
DivIVA localization in B. subtilis strains lacking certain lipids
wt
- PG
-PE
- CL
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes
Affinity for curvature = induces curvature
‘BAR domains as sensors or membrane curvature’
Peter et al., 2004, Science
Affinity for curvature = induces curvature
‘BAR domains as sensors or membrane curvature’
Peter et al., 2004, Science
Induction of curved membranes ?
liposomes + DivIVA
liposomes
200 nm
DivIVA
D
D
D
D
D
D
D
D
D
liposomes
Induction of curved membranes ?
200 nm
100 nm
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes
?
Does curvature really not play a role?
B. subtilis
E. coli
E. coli division mutant
MHD63
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes….., but not as we know it
Higher order DivIVA structures
‘Doggy bones’
Ø ~ 25 nm
Stahlberg, 2004, Mol. Mic.
( Cryo-negative stain EM )
?
~ 25 nm
Conceptual simplification:
Ø ~ 100 nm
?
‘Molecular Bridging’
1) self interaction (clustering) of subunits
2) subunits should be large (relative to curvature)
3) membrane interaction (weak)
- no other proteins / lipids / or curved proteins necessary -
Monte Carlo simulation
Monte Carlo simulation
Rules:
- cylinder 1 x 4 µm
- DivIVA oligomers (green) = spheres of 25 nm diameter
- curvature of membranes at transition from lateral wall to sides = diameter of 100 nm
- spheres can make max 8 contacts (doggy bone contains at least 8 DivIVA molecules)
- 2 membrane contacts maximal (based on our EM data)
- Epp and Epm in the range 1.5-6 k bT
(equivalent to 1-4 kcal/mol) ~in range of typical weak protein-protein attractions
- spheres can make 8 contacts
- 2 membrane contacts maximal
- spheres can make 4 contacts
- no limitations in membrane contacts
d = 50 nm
- No restrictions in
nr. of interactions
Epp = 2 k bT
Epm = 6 k bT
- 4 pp bonds
- membrane contact
= 1 pp contact
Epp = 2.5 k bT
Epm = 5.5 k bT
d =100 nm
d = 50 nm
- max 4 pp bonds
- membrane contact
= 2 pp contact
Epp = 3 k bT
Emp = 5.5 k bT
- max 6 pp bonds
- membrane contact
= 3 pp contacts
Epp = 3.5 k bT
Epm = 5.5 k bT
d =100 nm
-Max 8 pp bonds
-membrane contact
= 4 pp contacts
Epp = 3.5 k bT
Epm = 5.5 k bT
d = 50 nm
d =100 nm
Modelling of doggy bones
CBCB - Newcastle University
Rok Lenarcic
Ling Wu
Jeff Errington
Sven Halbedel
University of Oxford
Wouter de Jong
Loek Visser
Michael Shaw
University of Edinburgh
Davide Marenduzzo