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Structure and function of glutamate transporters from free energy simulations Turgut Baştuğ TOBB ETU

Transporter structures Transporters have larger structures, which are partly in and partly outside the membrane. Also they have no symmetries. Therefore they are much harder to crystallize First complete transporter structure: ABC (ATP binding cassette) transporter, Locher et al. 2002. First glutamate (aspartate) transporter: GltPh from Pyrococcus horikoshii, Gouaux et al. 2004; 2007) First sodium-potassium pump structure: Nissen et al. Dec. 2007) Prediction: Gouaux and Nissen will win the Nobel prize within a few years.

Transporters Structures & Functions Each time an action potential passes, Na and K concentration differences are depleted a little. To maintain the concentration differences, membrane proteins called sodium-potassium pumps work in the background, continuously pumping Na + and K + ions against their electrochemical gradient. In each cycle, it pumps 3 Na + out, 2 K + in, using 1 ATP molecule. Na-K pump is an example of primary active transporters which use the energy from ATP.

Glutamate transporters Glutamate is the major excitatory neurotransmitter in the central nervous system. Its extracellular concentration needs to be tightly controlled, which is achieved by glutamate transporters. They use the ionic gradients to transport 1 Glu in together with 3 Na + and 1 H + ions. Structure of a bacterial aspartate transporter (Gouaux et al. 2004) Each monomer in the trimer functions independently.

Structure of GltPh from Pyrococcus horikoshii Boudker, Ryan et al. 2007 Binding sites for Asp and two Na ions are observed.

MD simulations of the Asp transporter GltPh Crystal structure of GltPh gives information but incomplete MD simulations of GltPh reveal the binding site for the third Na ion, which was not observed in the crystal structure Complete characterization of the binding sites for the Na ions and Asp Binding free energy calculations for Na ions and Asp determine the binding order

Simulation system Protein Lipid Ions Water

The crystal structure is in closed state. After the Na Closed and open states of Gltph + ions and Asp are removed, the hairpin HP2 moves outward, exposing the binding sites. HP2

Opening of the extracellular gate HP2

Initial MD simulations of GltPh with 2 Na ions and Asp In the crystal structure, Na1 is coordinated by D405 side chain (2 O s) & carbonyls of G306, N310, N401 After (long) equilibration in MD simulations, D312 side chain swings 5 A and starts coordinating Na1, displacing G306 which moves out of the coordination shell. This picture is in conflict with the crystal structure. Proper question to ask: what is holding D312 side chain in that location in the crystal structure? The tip of the D312 side chain is the most likely site for Na3.

Movement of the D312 sidechain in MD simulations Initially, D312 (O) is > 7 A from Na1. After about 35 ns, it swings to the coordination shell of Na1, pushing away G306 (O) and also one of the D405 (O). This is conflict with the crystal structure.

Na ions in Gltph There are 2 Na ions and a ASP in the crystal structure of GltPh but experiments indicate presence of a third Na ion. Several suggestions have been made for the position of the third Na ion in the protein: Kavanaugh et al. Grewer et. al. and Tajkhorshid, This work supports Tajkhorshid 's suggestion for the position of the 3rd Na ion.

Hunt for the Na3 site after the experiments with radioactive Na + revealed its existence Reject those sites that do not involve D312 in the coordination of Na3 (Noskov et al, Kavanaugh et al.) Two prospective Na3 sites are found that involve D312 as well as T92 and N310 sidechains 1. In MD simulations that use the closed structure, the 5 th ligand is water. (Tajkhorshid, 2010) 2. In the open (TBOA bound) structure N310 sidechain is flipped around, which shifts the Na3 site, making the Y89 carbonyl as the 5 th ligand. (Question: Why isn t the Na3 site seen in the crystal structure?)

Comparison of Na3 sites from closed & open structures Na3 (closed structure) D312 (2), N310, T92, H 2 O (Huang and Tajkhorshid, 2010) Na3 (open structure) D312 (1), N310, T92, S93, Y89 (Our results)

Residues involved in the coordination of Na1 (Pair distribution functions for the Na O distances)

Ion Helix-residue Cryst. str. Closed state Open state Na3 TM3 T89 (O) 2.3 ± 0.1 2.3 ± 0.1 TM3 T92 (OH) 2.4 ± 0.1 2.4 ± 0.1 TM3 S93 (OH) 2.4 ± 0.1 2.3 ± 0.1 TM7 N310 (OD) 2.2 ± 0.1 2.2 ± 0.1 TM7 D312 (O 1 ) 2.1 ± 0.1 2.1 ± 0.1 TM7 D312 (O 2 ) 3.6 ± 0.2 3.5 ± 0.3 Na1 TM7 G306 (O) 2.8 2.4 ± 0.2 2.4 ± 0.2 TM7 N310 (O) 2.7 2.3 ± 0.1 2.4 ± 0.2 TM8 N401 (O) 2.7 2.4 ± 0.2 2.5 ± 0.2 TM8 D405 (O 1 ) 3.0 2.2 ± 0.1 2.2 ± 0.1 TM8 D405 (O 2 ) 2.8 2.2 ± 0.1 2.3 ± 0.1 H 2 O - 2.3 ± 0.1 2.3 ± 0.1 Na2 TM7 T308 (O) 2.6 2.3 ± 0.1 TM7 T308 (OH) 5.5 2.4 ± 0.1 HP2 S349 (O) 2.1 4.5 ± 0.3 HP2 I350 (O) 3.2 2.3 ± 0.1 HP2 T352 (O) 2.2 2.3 ± 0.1

Points to note Tl + ions are substituted for Na + ions in the crystal structure because they have six times more electrons and hence much easier to observe. Because Tl + ions are larger, the observed ion coordination distances are in general larger than those predicted for the Na + ions. For the same reason, some distortion of the binding sites can be expected (e.g. Na2) The path to the Na3 site goes through the Na1 site and is very narrow. Therefore Tl + substitution works for Na1 and Na2 but not for Na3. That is, the Na + ion at the Na3 site cannot be substituted by the Tl + ion at the Na1 site due to lack of space. This explains why the Na3 site is not observed in the crystal structure.

Coordination of Asp In the closed structure, Asp is coordinated by 10 N & O atoms (3 from HP1, 2 from HP2, 1 from TM7, 4 from TM8) In the open structure, HP2 gate opens, leading to loss of 2 contacts but another one is gained from TM8. In both cases, there is a 1-1 match between Exp. and MD. Asp stably binds to the open structure in the presence of Na3 and Na1. Removing Na1, destabilizes Asp which unbinds within a few ns. Corollary: Asp binds only after Na3 and Na1. Question: is there a coupling between Asp and Na1?

GltPh residues coordinating Asp Helix-residue Asp Cryst. str Closed state Open state Open (restr) HP1 R276 (O) a-n 2.4 3.0 ± 0.2 3.0 ± 0.2 3.0 ± 0.2 HP1 S278 (N) a-o 1 2.8 2.8 ± 0.1 2.8 ± 0.1 2.8 ± 0.1 HP1 S278 (OH) a-o 2 3.8 2.7 ± 0.1 2.8 ± 0.2 2.8 ± 0.1 TM7 T314 (OH) b-o 2 2.7 2.7 ± 0.1 2.8 ± 0.1 2.8 ± 0.1 HP2 V355 (O) a-n 2.9 2.9 ± 0.2 11.9 ± 0.4 11.9 ± 0.3 HP2 G359 (N) b-o 2 2.8 3.1 ± 0.2 6.1 ± 0.4 6.3 ± 0.3 TM8 D394(O 1 ) a-n 2.6 2.7 ± 0.1 2.7 ± 0.1 2.7 ± 0.1 TM8 R397(N 1 ) b-o 2 4.6 4.2 ± 0.2 2.7 ± 0.1 2.7 ± 0.1 TM8 R397(N 2 ) b-o 1 2.5 2.9 ± 0.2 2.9 ± 0.2 2.9 ± 0.2 TM8 T398(OH) a-n 3.2 3.2 ± 0.2 3.0 ± 0.2 3.0 ± 0.2 TM8 N401(ND) a-o 2 2.8 2.8 ± 0.1 3.0 ± 0.2 2.9 ± 0.2 In the open state HP2 gate moves away from Asp but it remains bound

Binding free energies for Na + ions and Asp in GltPh The crystal structure provides a snapshot of the ion and Asp bound configuration of the transporter protein but it does not tell us anything about the binding order and energies. We can answer these question by performing free energy calculations. The specific questions are: 1.We expect a Na + ion to bind first - does it occupy Na1 or Na3 site? 2.Does a second Na+ ion bind before Asp? 3.Are the binding energies consistent with experimental affinities? 4.Are the ion binding sites selective for Na + ions? 5.Can we explain the observed selectivity for Asp over Glu (there is no such selectivity in human Glu transporters) Once we answer these questions successfully in GltPh, we can construct a homology model for human Glu transporters and ask the same there.

FEP for Open/Closed Gltph FEP for Ions Ion in the bs is destroyed and a water created whereas a water in the bulk is destroyed and an ion is created. FE is calculated by TI scheme.

FEP for Open/Closed Gltph FEP for ASP ASP in the BS is destroyed and 5 water molecules are created whereas 5 water molecules in the bulk are destroyed and one ASP molecule is created. FE is calculated for chain A

Convergence of binding free energies in TI method TI calculation of the binding free energy of Na+ ion to the binding site 1 in Gltph. Integration is done using Gaussian quadrature with 7 points. Thick lines show the running averages, which flatten out as the data accumulate. Thin lines show averages over 50 ps blocks of data.

Na binding energies from free energy simulations Translocation free energy is obtained using free energy perturbation or thermodynamic integration. Free energy losses due to transl. and rotat. entropy are included (3 rd column). Binding free energies (in kcal/mol): Open structure Ion DG int DG tr DG b Na3-23.3 4.6-18.7 Na3-19.2 4.6-14.6 Na1-16.2 4.9-11.3 Na1 (Na3) -11.9 4.8-7.1 Closed structure Ion DG int DG tr DG b Na2-7.1 4.4-2.7 Na2-1.7 4.4 +2.7 Note that Na2 energy is positive, i.e. Na ion does not bind to Na2 (exp: -3.3)

Confirmation of the Na3 site from mutation experiments The T92A and S93A mutations reduce the experimental sodium affinities significantly relative to wild type (K 0.5 increases by x10). The same mutations reduce the calculated binding free energies at Na3 but not at Na1. (All energies are in kcal/mol) Wild type T92A S93A Na3-18.7 ± 1.2-11.2 ± 1.4-12.8 ± 1.2 Na1 (Na3) -7.1 ± 1.3-6.7 ± 1.2-6.4 ± 1.4 Conclusion: T92 and S93 are involved in the coordination of the Na3 site

Convergence of Asp binding free energy in TI method TI calculation of the binding free energy of Asp to the binding site in Gltph. Asp is substituted with 5 water molecules. First 400 ps data account for equilibration and the 1 ns of data are used in the production.

Asp binding energies (open structure) Contribution DG (kcal/mol) Notes Electrostatic -16.1-15.8 (FEP), -16.4 (TI) Lennard-Jones 4.6 3.8 (bb) + 0.8 (sc) Translational 3.3 Rotational 3.9 Conform. restraints 0.5 1.2 (bulk) - 0.7 (b.s.) Total -3.8 Forward and backward calculations agree within 1 kcal/mol (that is, no hysteresis) Convergence is checked from running averages Exp. binding free energy (-12 kcal/mol) includes gating & Na2 energy

Binding order from binding free energies The Na3 site has the lowest binding free energy, therefore it will be occupied first (-18.7 kcal/mol). Asp does not bind in the absence of Na1, hence Na1 will be occupied next (-7.1 kcal/mol). Asp binds after Na3 and Na1 (-3.8 kcal/mol). The HP2 gate closes after Asp binds. Na2 binds last following the closure of gate (-2.7 kcal/mol) Experiments confirm that a Na ion binds first and another one binds last but do not tell whether Asp binds after one or two Na ions. Presence of two Na ions obviously enhances binding of an Asp.

Lessons from the free energy simulations Correct reading of the crystal structure is essential: Respect the long and medium distance structure (e.g. the D312 side chain is correct). But be careful with short distance assignments of side chains (e.g. the N310 side chain has the wrong conformation in the closed structure). Free energy simulations can: help to resolve structural issues provide an overall picture for the binding processes confirm the reliability of the model via comparison with experimental binding free energies.

Conclusions Great deal of progress has been made in understanding the structure-function relations in ion channels. The field is now ripe for applications in biomedicine and pharmacology. Transporter structures have been solved more recently and much more work needs to be done to decipher the structure-function relations. New methods are needed to increase the simulation times to the milli to microsecond time domain.

People Serdar Kuyucak (Sydney Uni.) Germano Heinzelmann (Sydney Uni.) Murat Çavuş (Bozok Uni.)

Acknowlodgement Support from: TUBITAK ARC

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