A Look at the Structure and Mechanism of a Na+/H+ Antiporter

By Tiffany Tran

BackgroundNa+/H+ antiporters are integral membranes

Essential for regulations of pH, Na+ concentration, and cell volume in a cell

Important for cell viability and cell metabolism

Examples of how change in activity can affect a cell:Overactivation of NHE1 antiporter is harmful to heart

muscle cellsDeletion of antiporter in plants decrease salt tolerance

while overexpression creates salt-resistant plants

The Subject: NhaAMain Na+/H+ antiporter in Escherichia coli

and other enterobacteria

Uses electrochemical proton gradient to exchange Na+ for downhill flow of protons into the cell

Strictly regulated by pH change

Data collection and refinement statisticsNative 2 Native 1 SeMet SAD

Data Collectiona

Space group P212121 P212121 P212121

Cell dimensions a, b, c (Å) 108.9, 121.7, 123.6 108.8, 121.8, 124.7 109.9, 121.5, 124.1Wavelength 0.9686 0.9794 0.9717Resolution (Å)b 20-3.45 (3.57) 15-3.8 (3.93) 17-4.3 (4.45)Rmerge

b 5.0 (29.4) 6.1 (41.3) 8.3 (43.4)

I/sI b 30.5 (2.1) 17.8 (2.1) 23.1 (3.8)Completeness (%)b 91.7 (64.1) 98.1 (98.8) 99.7 (100)Redundancy 18.4 12.0 21.5

RefinementResolution (Å) 15-3.45No. reflections 19,993Rwork/ Rfree

c 30.1/31.6

No. atoms Protein 5,618B-factors Protein 121R.m.s deviations Bond lengths (Å) 0.01 Bond angles (º) 1.57

a The SeMet SAD data set was used for phasing, and the model was first constructed with data set Native 1 and subsequently refined to 3.45 Å resolution using the data set Native 2. A single crystal was used for each data set. b Highest resolution shell is shown in parenthesis. c 2.9 % of the data were randomly excluded from the refinement to calculate Rfree.

•At acidic pH, NhaA is downregulated and crystals formation is more ordered

•This allows for easier structure and architecture analysis

Overall Architecture388 amino acids with both terminus exposed to

cytoplasm12 transmembrane segments (TMSs)Molecule was 45 Å x 40 Å with height was 50 ÅTMSs III and X are S-shaped helices, helix IX is bent,

and helices VII and VIII are extraordinarily shortTMSs IV and XI are of opposite orientation consists

of short helix, polypeptide chain, and short helixThey form an assemblyUnfavorable for polar ends to be in the core of the

membrane but Asp 133 and Lys 300 acts as a charge compensation where the extended chain crosses

Architecture cont.Periplasmic face is flat due to

anti-parallel, double stranded β sheet that lays parallel to the membrane

Cytoplasmic face is rough and rigid due to several helices protruding and flexible loops

Two domains:Domain A: two bundles of three

helices (III, IV, V; X, XI, XII)Domain B: linear bundle of six

helices (I, II, VI, VII, VIII, IX)

The FunnelIn the center of

domain interface, two funnels are observed: one open to the cytoplasm, one open to the periplasm

Funnel is blocked in the crossing of TMSs IV/XI assembly

Funnel does not form continuous pore

Substrate Binding at Acidic pH-locked Conformation The negatively charged lining of the funnel attracts cations from the

cytoplasmic sideFunnel narrows so large hydrated ions cannot continue downAt the end of the funnel, there is Asp 164 which suggests binding site

Several other residues contribute to binding site (Asp 163, Asp 133, Thr 132)

16 Å away from Asp 164, Asp 65 lies at the tip of the shallow funnel facing the periplasm

A periplasmic barrier is formed by non-polar residues around helices II, IVp, and XIp

Mechanism of pH-regulated Na+/H+ exchange NhaA is pH dependent: activity

alters at three orders of magnitude between pH 7 and 8 and is fully downregulated below pH 6.5

Questions asked:Where is the ‘pH sensor’?What is the structural element that

transmits and converts the pH signal into a change in activity?

How is the change accomplished?

“pH Sensor”Mutagenesis of loop of helices VIII-IX

drastically changes pH dependence of NhaAConformational change induced by alkaline pH

is observed at N terminus of helix IXThe pH dependence of these conformational

changes at the cytoplasmic side parallels the pH dependence of NhaA activation

Therefore a “pH sensor” is located at entrance of cytoplasmic passageway

pH signal elicits conformational change which is transmitted to activate NhaA

“pH Sensor” cont.Helix IX is suggested to

be most likely to transmit pH signal because of its flexibility for long-range conformational change

N-terminus of the helix undergoes pH-induced change

Kink at the center is in close proximity of the helices IV/IX assembly

Mechanism This transporter uses the alternating access mechanism Binding site either faces inwards or outwards

Interconversion only possible when substrate is bound Binding sites are two aspartates on helix V which cannot go through a

conformational change TMSs IV/XI assembly is most suitable for fast conformational change Therefore it is concluded that TMSs IV/XI assembly allows for alternating

access of substrate-binding site

The ExchangeAt alkaline pH, pH signal causes conformational

change in helix IX which causes reorientation of helices IVc and XIpExposes binding site (Asp 164, Asp 163, and possibly Thr

132)Removes periplasmic barrier

Once Na+/Li+ binds, it causes charge imbalance that shifts XIp and IVc so that the binding site faces the periplasm

On release of the ions, they are hydratedBoth aspartates are protonated and a conformational

change causes the binding sites to face the cytoplasm Deprotonation completes a cycle

The Exchange cont.NhaA is reversible, so direction of exchange

depends on electrochemical potential difference of Na+/Li+ compared to H+

The rate of exchange depends on movement of extended chains of helices IVc and XIp in response to a change in pH

Because of NhaA’s unique TMSs IV/XI assembly, it is one of the fastest transporters (89,000 turnovers per minute)

ConclusionNa+/H+ exchange occurs in a funnel that is

formed in the antiporter“pH sensor” triggers conformational change

in the protein and activates itTMSs IV/XI assembly is a unique and

important key to substrate bindingInduced by pH, NhaA exchanges one Na+ for

two H+ to regulate cell viability

ReferencesHunte, Carola, Emanuela Screpanti, Miro

Venturi, Abraham Rimon, Etana Padan, and Hartmut Michel. "Structure of a Na+/H+ Antiporter and Insights into Mechanism of Action and Regulation by PH." Nature (2005): 1197-202. 

http://www.nature.com/nature/journal/v435/n7046/full/nature03692.html