SLIDE SHOW IWild-type mechanism

K319

-

-+

-+

C

E325

H322

D240R

E126

148144

IV

VIIV

-E269

VIII

+R302

IX

X

A. In the ground state the permease is protonated

H+

K319

-

+

-+

C

E325

H322

D240R148144 -

E126

IV

VIIV

-E269

VIII

+R302

IX

X

B. Substrate binds from the outside

H+o

So

K319

-

-+

-+

C

E325

H322

D240R

E126

148144

IV

VIIV

-E269

VIII

+R302

IX

X

So

C. Binding of substrate causes a conformational change disrupting the interaction of Glu269 and His322 and ….

H+

+

-+CR

R302

E126

148144

IV

VIIV

IX

-

D240

-

E 325

H 322

K 319

+

X

-

E269

VIII

Si

D. ... causes substrate to become accessible to the internal surface, and the proton moves to His322/Glu325.

H+

+

-+CR

R302

E126

148144

IV

VIIV

IX

-

D240

-

E 325

H 322

K 319

+

X

-

E269

VIII

Si

E. Rotation of Helix X continues towards the hydrophobic phase of the membrane,

H+

+

-+CR

R302

E126

148144

IV

VIIV

IX

-

D240

-E 325H

322K

319

+

X

-

E269

VIII

Si

F. raising the pKa of Glu325.

H+

+

+CR

R302

148144

IV

V

IX

-

-

E126

VIID240

-E 325H

322K

319

+

X

-

E269

VIII

G. Substrate dissociates to the inside,

H+

Si

Si

H. And Glu325 re-juxtaposes with Arg302.

+

-+CR

R302

E126

148144

IV

VIIV

IX

-

D240

-E 325H

322K

319

+

X

-

E269

VIII

H+

K319

-

+

-+

C

E325

H322

D240R148144 -

E126

IV

VIIV

-E269

VIII

+R302

IX

X

I. The proton is released to the inside,

H+i

K319

-

-+

-+

C

E325

H322

D240R

E126

148144

IV

VIIV

-E269

VIII

+R302

IX

X

J. the permease relaxes into the ground state and becomes protonated from the outside.

H+o

N7C5

C5

_+ + + +

_ _ _

MT

S-1

-MT

S

MT

S-3

-MT

S

o-P

DM

p-P

DM

TDGno

lin

ker

K. Crosslinking between Asp240 (helix VII) and Lys319 (helix X): Effect of ligand binding

MTS-1-MTS: 3 ÅMTS-3-MTS: 5 Å; flexibleo-PDM: 6 Å; rigidp-PDM: 10 Å; rigid