Structure and function of sodium-proton antiporters
File(s)
Author(s)
Lee, Chiara Natasha
Type
Thesis
Abstract
Sodium-proton (Na+/H+) antiporters are secondary membrane protein transporters
present in all living cells and are critical for sodium, pH and cell volume homeostasis.
Their deregulation of transport activity has been linked to human diseases, such as,
hypertension, heart failure and epilepsy and consequently they may be targets for
drugs. In 2005, the first crystal structure of a Na+/H+ antiporter, NhaA from
Escherichia coli, was solved at 3.45 Å resolution in an inward-facing conformation at
pH 4 where the protein is inactive. Like many Na+/H+ antiporters, the activity of
NhaA is regulated by pH and is active above pH 6.5.
The main goal of this thesis was to solve a crystal structure of a Na+ /H+ antiporter in
an active state. One of the problems in producing milligram quantities of purified
membrane protein for crystallography is poor overexpression. For this reason, we first
sought to improve membrane protein overexpression by developing a new expression
platform, which we have called “MemStar” in E. coli using a test-case of control
proteins, overall showing a boost in expression levels to at least 12 mg.L-1.
This thesis describes the crystal structure of NapA from Thermus thermophilus, an
NhaA homologue, which was solved to 3 Å in an outward-facing conformation at pH
7.8 in an active state. This NhaA homologue was selected as purified protein could
grow better diffracting crystals than NhaA. The stability of NapA was also more
suitable for purification in a small micelle detergent to improve diffracting resolution.
Although NhaA crystals did not form above pH 6.5, a stabilised mutant was useful to
confirm the position of a critical residue important in the mechanism.
Structural comparisons with the NapA structure show the core domain moving
relative to the dimerisation domain, similar to a rocking bundle model observed in
other structures of different secondary active transporters sharing conserved structural
features in their membrane protein folds also present in NhaA and NapA. This work
has provided us with a fresh insight into the mechanism of Na+/H+ antiporters.
present in all living cells and are critical for sodium, pH and cell volume homeostasis.
Their deregulation of transport activity has been linked to human diseases, such as,
hypertension, heart failure and epilepsy and consequently they may be targets for
drugs. In 2005, the first crystal structure of a Na+/H+ antiporter, NhaA from
Escherichia coli, was solved at 3.45 Å resolution in an inward-facing conformation at
pH 4 where the protein is inactive. Like many Na+/H+ antiporters, the activity of
NhaA is regulated by pH and is active above pH 6.5.
The main goal of this thesis was to solve a crystal structure of a Na+ /H+ antiporter in
an active state. One of the problems in producing milligram quantities of purified
membrane protein for crystallography is poor overexpression. For this reason, we first
sought to improve membrane protein overexpression by developing a new expression
platform, which we have called “MemStar” in E. coli using a test-case of control
proteins, overall showing a boost in expression levels to at least 12 mg.L-1.
This thesis describes the crystal structure of NapA from Thermus thermophilus, an
NhaA homologue, which was solved to 3 Å in an outward-facing conformation at pH
7.8 in an active state. This NhaA homologue was selected as purified protein could
grow better diffracting crystals than NhaA. The stability of NapA was also more
suitable for purification in a small micelle detergent to improve diffracting resolution.
Although NhaA crystals did not form above pH 6.5, a stabilised mutant was useful to
confirm the position of a critical residue important in the mechanism.
Structural comparisons with the NapA structure show the core domain moving
relative to the dimerisation domain, similar to a rocking bundle model observed in
other structures of different secondary active transporters sharing conserved structural
features in their membrane protein folds also present in NhaA and NapA. This work
has provided us with a fresh insight into the mechanism of Na+/H+ antiporters.
Version
Open Access
Date Issued
2013-03
Date Awarded
2013-07
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Drew, David
Iwata, So
Cameron, Alexander
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)