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

coiled coil prediction by LUPAS

secondary structure prediction by PSIPRED

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

DG = DivIVA-GFP

V = membrane vesicles

Lip = liposomes

D = DivIVA

G = GFP

Binding to another (membrane) protein?

70 %

20 %

30 %

membrane vesicles

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 - CL-PE

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 liposomes + DivIVA

DDDDD

DDDD

liposomes

DivIVA

200 nm

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

Stahlberg, 2004, Mol. Mic.

( Cryo-negative stain EM )

‘Doggy bones’Ø ~ 25 nm

Ø ~ 100 nm

~ 25 nm

?

?

Conceptual simplification:

1) self interaction (clustering) of subunits

2) subunits should be large (relative to curvature)

3) membrane interaction (weak)

‘Molecular Bridging’

- 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 d =100 nm

- No restrictions in nr. of interactions

Epp = 2 k bTEpm = 6 k bT

- 4 pp bonds- membrane contact = 1 pp contact

Epp = 2.5 k bTEpm = 5.5 k bT

d = 50 nm d =100 nm

- max 4 pp bonds- membrane contact = 2 pp contact

Epp = 3 k bTEmp = 5.5 k bT

- max 6 pp bonds- membrane contact= 3 pp contacts

Epp = 3.5 k bTEpm = 5.5 k bT

d = 50 nm d =100 nm

-Max 8 pp bonds-membrane contact= 4 pp contacts

Epp = 3.5 k bTEpm = 5.5 k bT

Modelling of doggy bones