Cartoon model of alternating access in
Mhp1. The thin gates (blue, TM10 and TM5) control access
from the outside and inside to the central binding site. The thick
gate is formed by the hash motif (yellow, TM3+TM8
and TM4+TM9). It rotates on an axis roughly parallel to TM3 relative
to the bundle (red) and controls the permeation across the
membrane. (This is a flash animation. Right-click and select
Play to restart it.)
Secondary transporters couple the free energy stored in an ionic
gradient to the movement of solutes across the cell membrane. The
coupling enables these transmembrane proteins to transport small
molecules against their own concentration gradients. The transporters
function by cycling between different conformational states in which
access to the central binding site is switched from the extracellular
solution to the intracellular compartment. This abstract
alternating access model was proposed by Oleg
Jardetzky in 1966 (Nature 211 (1966), 969–970) but its
structural basis was not established for any transporter family.
According to the model, a cation-substrate symporter would go
through a cycle of a number of distinct conformations:
In the outward-facing open conformation, the substrate
and ions can enter the binding sites from the extracellular space. The
binding site is located near the centre of the protein.
A conformational change takes place that closes access to the
outside. Ion and solute are sealed off in this occluded
Then protein switches to the inward-facing open
conformation. The binding sites become connected to the intracellular
compartment and ion and solute are released.
The cycle is closed by a transition back to 1 in the absence of
substrate. Whereas the transitions 1→2 and 2→3 are coupled
to the ionic gradient's free energy, the transition 3→1 must be
able to occur spontaneously through activation by thermal
We studied the hydantoin permease from Microbacterium
liquefaciens, a sodium-coupled nucleobase symporter and a member
of the NCS1
family. Mhp1 shares the same general architecture with other
transporters such as the sodium-amino acid transporter
LeuTaa (which is a bacterial homolog of the important
mammalian neurotransmitter:sodium symporter (NSS) family). Weyand
et al. solved the two outward-facing conformations of Mhp1 in
2008. In 2010 we determined the inward facing conformation (Shimamura et al (2010)) and
carried out computer simulations of Mhp1 in all three
conformations. Using a novel simulation method (dynamic importance sampling)
we were also able to simulate the conformational transition itself.
Based on the combination of structural data and simulations (movie
in MPEG4 format) we could identify the moving elements of the
transport mechanism and propose a sequence of events that leads to
solute transport as shown in the animated cartoon.
Our hypothetical model for secondary transport in Mhp1 contains the
following steps and describes the transporter as a triple-gated
In equilibrium and in the absence of substrate and ions, the
protein switches spontanously between the inward facing and the
outward facing state.
Once Na+ binds to the outward facing state, it
stabilizes it and allows the substrate (here benzyl-hydantoin) to
The extracellular thin gate closes.
The thick gate switches from outward facing to inward
The intracellular thin gate opens and substrates are released to
the intracellular space.
Go back to 1.
By utilizing a system of (synchronized) gates, the protein appears
to prevent the formation of a continuous conduction pathway, which
would lead to uncoupled leakage of Na+ into the cell or
loss of substrate out of the cell. In other words, our model describes
in molecular detail a transport process that has the properties
required by the alternating access mechanism.
Accessibility of the binding sites of
Mhp1 in three conformational states. Computer simulations of
the Mhp1 transport protein (helices in cartoon representation, front
cut away) in a lipid membrane (gray) were carried out to determine the
functional states of three crystallographic structures of Mhp1 (A:
outward facing open, 2JLN;
B: outward facing occluded, 2JLO,
and C: inward facing open 2X79). Water
molecules (red/white) can only access the substrate and sodium binding
site in the open conformations, and the binding sites are sealed in
the occluded state. This is the behaviour is predicted by the
alternating access mechanism. Repeated simulations showed that
inward-facing conformation (C) readily released the bound
Na+ ion; the positions of the ion (magenta spheres) from
six simulations were all overlaid in panel C and define the exit