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Published online before print December 2, 2004, 10.1110/ps.04973805
Protein Science (2005), 14:183-192. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Computational analysis of the transient movement of helices in sensory rhodopsin II

Y. Sato1, M. Hata, S. Neya and T. Hoshino

Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan

Reprint requests to: T. Hoshino, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan; e-mail: hoshi{at}p.chiba-u.ac.jp; fax: +81-43-290-2925.

(RECEIVED July 6, 2004; FINAL REVISION September 10, 2004; ACCEPTED September 13, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
MD simulation of sensory rhodopsin II was executed for three intermediates (ground-state, K-state, M-state) appearing in its photocycle. We observed a large displacement of the cytoplasmic side of helixF only in M-state among the three intermediates. This displacement was transmitted to TM2, and the cytoplasmic side of TM2 rotated clockwise. These transient movements are in agreement with the results of an EPR experiment. That is, the early stage of signal transduction in a sRII–HtrII complex was successfully reproduced by the in silico MD simulation. By analyzing the structure of the sRII–HtrII complex, the following findings about the photocycle of sRII were obtained: (1) The hydrogen bonds between helixF and other helices determine the direction of the movement of helixF; (2) three amino acids (Arg162, Thr189, Tyr199) are essential for sRII–HtrII binding and contribute to the motion transfer from sRII to HtrII; (3) after the isomerization of retinal, a major conformational change of retinal was caused by proton transfer from Schiff base to Asp75, which, in turn, triggers the steric collision of retinal with Trp171. This is the main reason for the movement of the cytoplasmic side of helixF.

Keywords: sensory rhodopsin II; molecular dynamics simulation; signal transduction; transducer; movement of helixF

Abbreviations: sRII, sensory rhodopsin II • HtrII, transducer molecule • TM, transmembrane • BR, bacteriorhodopsin • MD, molecular dynamics • RMSD, root mean square deviation • VDW, van der Waals

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04973805.


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Sensory rhodopsin II (sRII) is a membrane protein that functions as a sensor for phototactic avoidance of blue-green light in the cell membrane of Natronobacterium pharaonis (Takahashi et al. 1985, 1990). The transmission of a photosignal from the photoreceptor sRII to the following cytoplasmic signal cascade is achieved by a transducer molecule that binds tightly and specifically to its cognate receptor (Zhang et al. 1999). This transducer molecule consists of two transmembrane helices (TM1 and TM2). The function of sRII is activated through a cyclic process initiated by the absorption of a photon. The photocycle is characterized by a series of intermediates, sRII498, K540, L488, M390, and O560, in which the subscripts denote the wave-lengths of the respective absorption maxima (Kamo et al. 2001).

The photocycle of sRII is effectuated by a retinal chromophore that is bound to the Lys205 residue through a Schiff-base linkage. Retinal undergoes an electronic excitation and shows photochemical transformation. In sRII498, retinal is in the all-trans configuration. A photon triggers an isomerization of retinal and leads to the transition sRII498->K540.The intermediate K540, which contains a strained 13-cis configuration of retinal, thermally decays to L488 (Kandori et al. 2001).

During the L488->M390 transition, the proton bound to retinal’s Schiff base is transferred to Asp75 (Sasaki and Spudich 1999; Furutani et al. 2002). It was clarified by the electron paramagnetic resonance spectroscopy experiment that the outward movement of cytoplasmic side of helixF occurs before M-state (Wegener et al. 2000). The movement of helixF triggers a rotation of its transducer (TM2) (Klare et al. 2004), and its transformation is converted to the cytoplasmic cellular signal. Recently, the X-ray structure of the complex of Natronobacterium pharaonis sRII with the receptor-binding domain of HtrII was obtained at 1.93 Å resolution (Gordeliy et al. 2002), and it provided an atomic picture of the first step of the signal transduction.

Molecular dynamics simulation allows observation of the dynamic fluctuation of the protein structure with a femto-second timescale and at an atomic level. This is an advantage of computer simulation from other experimental methods. In our previous ab initio computational study about BR (Murata et al. 2000, 2002, 2003), which is the same bacterial retinal protein, we proposed the importance of the internal water chain for the proton pump activity. These internal water molecules are often difficult to detect by experimental methods such as crystallographic study. The validity of this proposal has been proved in some other groups’ studies (Schobert et al. 2003; Lee 2004). Similarly, the molecular dynamics simulation on the sRII–HtrII complex is expected to clarify the mechanism of signal transduction more clearly. In this study, we performed molecular dynamics simulations of the three intermediates (ground-state, K-state, M-state) in sRII’s photocycle, and observed the motion of atoms when sRII transmits the signal to the transducer (HtrII).


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
We simulated Natronobacterium pharaonis sRII in the three different intermediates. In the simulation, the atoms constituting the lipid bilayer and water molecules were included along with the sRII and HtrII protein. Figure 1Go shows the simulation model for this sRII–HtrII, lipid-bilayer, and water-molecule system. More details are provided in Materials and Methods. In the following, we report several findings about the signal transduction in the sRII–HtrII complex deduced from the present computer simulations.



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  		Figure 1. Simulation model for sRII–HtrII–lipid layer–solvent water system. (A) sRII–HtrII complex manually inserted into the cavity of lipid bilayer. (B) Structure of a component of the lipid bilayer:PGP-Me. PGP-Me is the main component of purple membrane (PM). (C) The model containing the water molecules at the top and bottom sides of the lipid–protein complex. (D) The lipid–protein complex viewed from the cytoplasmic side. Waters are not shown for the sake of visual clarity.

 
RMSD, thickness of lipid bilayer, and retinal surroundings in three intermediates
Firstly, root mean square deviation (RMSD) from the energy-minimized structure was examined in three intermediates simulations, respectively. The RMSD value was extracted only from the backbone atoms of the sRII–HtrII complex to neglect the minor fluctuation of side chains. In the respective simulation, RMSD value rapidly changed in the heating calculation for the first 60 psec, and was kept almost constant during the following 500-psec simulation at 310 K. The average RMSD value during the M-state simulation (0–500 psec) is 1.7 Å, the largest among the three intermediates. The next simulation of the K-state (1.5 Å), and the smallest is the ground-state simulation (0.8 Å).

Next, the change of the thickness of lipid bilayer along the total of 560-psec simulation was examined (Fig. 2Go). The thickness is defined as the distance between the average z-position of oxygen atoms in the hydroxyl group of the glycerol located at the cytoplasmic side and the average z-position of oxygen atoms at the extracellular side; each of these oxygen atoms makes an ester bond to the fatty-acid chains in lipid molecules (PGP-Me). This value gradually decreased with the progress of the simulation, mainly in the first 60-psec heating simulation, and approached a constant value. The averaged thickness value for the last 200 psec (300–500 psec) of the simulation is 31.61 Å, and is consistent with the experimentally measured thickness by electron diffraction of Harobacterium salinarium (Mitsuoka et al. 1999). This indicates that the present simulation for a lipid-protein model system successfully reproduces the physiological condition.



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  		Figure 2. The fluctuation of the thickness of lipid bilayer (the average distance between the hydroxyl group’s oxygen atoms of glycerol) during the equilibrating and the subsequent dynamics simulations in the ground-state. Dashed line shows the value of the thickness obtained by the electron diffraction experiment in Halobacterium salinarium (Mitsuoka et al. 1999).

 
We also analyzed the change of the interaction of the retinal with the surrounding amino acid residues following the retinal photoisomerization. Figure 3Go shows the change of the distances between Schiff base and two amino acid side chains as a function of simulation time. One notable binding factor is the hydrogen bond between the Schiff base and the hydroxyl oxygen atom of Thr 79 (Fig. 3AGo), and the other is the hydrogen bond between Schiff base and the calboxyl oxygen atom of Asp201 (Fig. 3BGo). Asp201 has two equivalent calboxyl oxygen atoms in its side chain, but we focus only on the one nearest to the Schiff base. The distance between the Schiff base and the hydroxyl oxygen of Thr79 was close in K-state and ground-state, but more distant in M-state. On the other hand, the distance between the Schiff base and the calboxyl oxygen atom of Asp201 was close in K-state compared with the other two intermediates. In K-state only, the Schiff base made the hydrogen bonds with the hydroxyl oxygen atom of Thr79 as well as the calboxyl oxygen atom of Asp201; these hydrogen bonds disappeared in M-state. According to the changes in these hydrogen-bond interactions and the retinal conformation, we summarize the pattern of chromophore–protein interaction in the three intermediates as follows: In the ground state, the retinal has an all-trans conformation, and the Schiff-base proton faces the direction of the extracellular region. The retinal stably interacts with hydroxyl oxygen atom of Thr79 in the extracellular region. After retinal photoisomerization, retinal has the strained 13-cis conformation, and the Schiff-base proton turns to the reverse direction, that is, the opposite of the extracellular region. In spite of this conformational change, the Schiff-base proton still interacts with two residues in the extracellular region. This Schiff-base protonation state is fairly unstable, and would result in the proton release to Asp75 in the extracellular region. In M-state, the interaction with the extracellular region disappears due to the Schiff-base proton release to Asp75, the instable chromophore–protein interaction is cancelled, and the constraint retinal structure recovers to the stable form.



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  		Figure 3. Distance between Schiff base and two amino acid residues as a function of simulation time in three intermediates. (A) The distance between Schiff base and oxygen atom of the hydroxyl side chain of Thr79. (B) The distance between Schiff base and the oxygen atom of the calboxyl side chain of Asp201.

 
Interhelical hydrogen bonds from helixF
Interhelical hydrogen bonding was surveyed between helixF and the other helices of sRII, using the data of the ground-state simulation. The criteria for keeping a hydrogen bond with the donor – H – acceptor formation is as follows: (1) within 4 Å for the distance between donor and acceptor atoms; (2) within 60° for the H – donor – acceptor angle. Table 1Go shows interhelical hydrogen bonds that meet the criteria for >30% of the time in the 500-psec simulation. In total, 12 hydrogen bonds were detected between helixF and other helices. In nine cases, the amino acid residues of helixF are in the donor character, and in three cases, the amino acids of helixF are acceptors. Six hydrogen bonds are located in the cytoplasmic half side, and the remaining six bonds were in the extracellular half side. The total number of the amino acid residues participating in these hydrogen bonds is 13, and eight amino acid residues of these are conserved in the corresponding sequence of BR (Halobacterium salinarium). If the carbonyl group of the main chain is also taken into account for hydrogen bonds, almost all of the amino acid residues participating in the interhelical hydrogen bonds are conserved in BR. This indicates that these hydrogen bonds are essential for both BR and sRII. The significant common features between them are the presence of seven helices and the experimentally observed motion of the cytoplasmic half side of helixF (Wegener et al. 2000). Furthermore, all six hydrogen bonds appearing in the cytoplasmic half side are between helixF and helixE. On the other hand, the counterpart of the hydrogen bond from helixF are various (helixC, helixD, helixE, and helixG) in the extracellular half side (Fig. 4Go). That is, the extracellular side of helixF has a strong interaction with the other six helices, while the cytoplasmic side is connected only in the one direction. This enables the movement of the cytoplasmic side of helixF perpendicular to the direction of helixE.


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  		Table 1. Hydrogen bonds between helixF and other helices of sRII
 


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  		Figure 4. Sketch of the hydrogen bond network between helixF and other helices of sRII viewed from the cytoplasmic side. The thick arrow is the direction of the movement of helixF proposed by EPR experiments (Wegener et al. 2000).

 
The movement of helixF
To clarify the mechanism for the movement of helixF, the dynamics of three intermediates (ground-state, K-state, and M-state) were compared from the simulation data. First, we measured the fluctuation of the distance between the center of whole protein (sRII) and the center of the cytoplasmic half side of helixF that consisted of 13 residues (residues 158–170). The distances are presented in Figure 5Go along the simulation time.



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  		Figure 5. Distance between the center of sRII (residues 1–225) and the center of cytoplasmic half side (residues 158–170) in the course of the simulation.

 
The distance in M-state is the largest among the three simulations, and the fluctuation is also large in M-state. This means that the cytoplasmic half side of helixF moves in the direction away from the center of sRII when sRII is in M-state. The distance in K-state is larger than that in the ground-state, but the deviation from the ground-state gradually became small as the simulation progressed. Figure 6Go shows the superimposed view of the average structures for the last 50 psec in the ground-state and M-state simulations. It is confirmed from this figure that the cytoplasmic half side of helixF indeed moved outward from the protein, and the cytoplasmic side of TM2 also moved in accordance with this movement. This movement is consistent with the result obtained by EPR experiment by Wegener et al. (2000).



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  		Figure 6. Superimposed structure of M-state and the ground-state. Each structure is the average of the last 50 psec of the simulation. A green ball represents the center of whole protein, and the red and blue balls represent the center of the cytoplasmic side (residues 158–170) of helixF. Arrow shows the direction of movement of helixF in M-state.

 
To clarify the driving force of this movement, we measured the van der Waals (VDW) interaction energy between retinal and each amino acid residue of sRII, both in the ground state and M-state simulations (Fig. 7AGo). Seven peaks appear to be in the energy, that is, the interacting residues are divided into seven groups. These groups match the seven helices of sRII, and helixC and helixF especially have a strong interaction with retinal. Alignment of the amino acid sequence of 16 bacterial retinal proteins also reveals a high degree of sequence conservation in helixC and helixF (Fig. 7BGo). Hence, these helices would mainly contribute to the retinal binding to the sRII, or have a significant influence on the retinal structural change. Then, the energy difference between the ground state and M-state is shown in Figure 7CGo. This clearly indicates that the interaction between retinal and Trp171 dramatically changed and became less stable in M-state than in the ground-state. We speculate that the isomerization of retinal results in this energetic instability, and the recovery of this instability causes the movement of helixF.



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  		Figure 7. (A) van der Waals interaction energy between retinal and each residue of sRII. (B) Degree of sequence conservation among 16 bacterial retinal–proteins (bR, hR, sRI, sRII). First, we aligned the amino acid sequence of the 16 retinal proteins, then determined the degree of amino acid sequence conservation of Natronobacterium pharaonis sRII. The degree is calculated from the following equation: degree = (the number of proteins in which sRII sequence is conserved at the corresponding position/total number of bacterial retinal proteins). (C) Difference of van der Waals interaction energy between M-state and the ground-state. The negative value means that the interaction becomes weaker in M-state than the ground-state.

 
The large movement of helixF in M-state, however, cannot be explained only by the isomerization of retinal, because K-state also has a 13-cis retinal structure. Hence, we compared the structure of M-state with K-state to examine the factor that enhances the retinal–Trp171 interaction other than the isomerization of retinal. The major difference between K-state and M-state is the protonation state. In the L->M conversion, the proton bound to the Schiff base transfers to the Asp75 (Sasaki and Spudich 1999), and the charging state of the Schiff base is converted from the positive to the neutral. Figure 8Go shows the structures around the retinal both in K-state and in M-state, in which two structures are superimposed with respect to the backbone atoms of whole protein (sRII) and each structure is the average of the last 50 psec of the simulation. In K-state, the retinal is pulled in the direction of Thr79, because there appears to be a hydrogen-bond connection between the Schiff base and Thr79 due to the positive charge at the Schiff base. Asp75 further supports the approach of Thr79 to the retinal by making a hydrogen-bond network through Schiff base–Thr79–Asp75, because Asp75 is negatively charged in K-state. It should be emphasized that the retinal has a strained structure in K-state. On the other hand, the retinal is released away from Thr79 in M-state, because the hydrogen-bond connection with Thr79 is canceled due to the neutral charge at the Schiff base. Since Asp75 also becomes neutral, the hydrogen-bond network through Schiff base–Thr79–Asp75 disappears in M-state. The distance between the nitrogen atom of Schiff base and the hydroxyl oxygen atom of Thr79 during the last 50 psec of simulation was 3.25 Å in K-state and 3.39 Å in M-state. This release of retinal from Thr79 causes the positional shift of retinal, which induces the steric collision of the retinal with Trp171. This collision would result in the movement of helixF in M-state.



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  		Figure 8. Superimposition of atom geometry between M-state and K-state. Each geometry was obtained from the average structure during the last 50 psec of the MD simulation. M-state is represented by a ball-and-stick, and K-state structure is represented by a thin stick.

 
Spudich et al. (1997) reported that the D73N mutation of Halobacterium salinarum sRII causes the constitutive signaling in its unphotostimulated state. Asp73 in Halobacterium salinarum sRII corresponds to Asp75 in Natronobacterium pharaonis sRII, and both aspartic acids may have the same role. From our structural investigation, it is clarified that this hydrogen-bond network (Schiff base->Thr79->Asp75) controls the conformation of retinal, and consequently, regulates the movement of helixF. The residues participating in this extracellular hydrogen-bond network from Schiff base and interacting with retinal are highly conserved among retinal proteins of various species. It was reported that the outward movement of helixF also occurred in BR (Koch et al. 1991; Subramaniam 1993). From these two facts, it is speculated that the movement of helixF is seen in other retinal proteins, and the mechanism is common among them, although each of these retinal proteins has a respective different function. Further, we speculate that sRII’s function of signal transduction is induced by the ability of binding to the cognate transducer (HtrII), and BR does not have the binding ability to HtrII.

Signal transmission to HtrII
To investigate how the signal transmits to the receptor (HtrII), we executed an additional 1.5-nsec MD simulation in M-state because a large movement of helixF had been observed. Accordingly, a total of 2 nsec of MD simulation was performed for the M-state, and the structural change of the transducer (HtrII) was examined in detail. Figure 9AGo shows the conformational change of TM2 and the rotational angle of three amino acid residues (Ala79, Ala80, Thr81) in the course of MD simulation. For determining the axis of TM2, we separated the amino acid residues in TM2 into two parts: extracellular (residues 54–67) and cytoplasmic areas (residues 68–82), and calculated the center point of each area. We defined the axis of TM2 as a vector connecting these two points, and calculated the rotational angle of three amino acid residues from the initial structure. The cytoplasmic side of TM2 started to rotate clockwise at 1 nsec, followed by the appearance of a maximum rotational displacement at 1.8 nsec. This rotational movement of TM2 is consistent with the movement detected by the electron paramagnetic resonance experiment (Wegener et al. 2001). The degree of rotational movement is shown in Figure 9BGo. Three amino acid residues (Ala79, Ala80, Thr81) are located in midst of the cytoplasmic side of TM2. The movements of the side chain of these three amino residues are in the range of ~2–3 Å, and the average value of the rotational angle of three amino acid residues is 24.8°. The motion observed by the EPR experiment (Wegener et al. 2001) was also in the same range. In our simulation, the rotational movement was observed in the time scale of 1 nsec, which suggests that the signal transduction to HtrII occurs in a considerably short time. In this study, the first signal-transduction step has been successfully reproduced in silico. It is, however, still unclear how this TM2 transition is converted into a cellular signal. We are planning to observe this cellular signal by executing MD simulation with a larger model system, including the cytoplasmic inner side of proteins in the future.



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  		Figure 9. (A) Conformational changes of cytoplasmic side of TM2 in the course of a 2-nsec MD simulation, viewed from the cytoplasmic side. Each structure was superimposed on the initial structure of M-state MD simulation. Red balls represent C{alpha} carbon atoms of the three amino acid residues (Ala79, Ala80, Thr81) of TM2 in the initial structure. Blue balls represent those in the snapshot structure at each simulation time. Rotational angle of each amino acid residue from the initial structure is shown below the snapshot structure at the respective simulation time. (B) Side-chain locations of these three amino acid residues. The initial structure is represented by sticks and the snapshot structure at 1800 psec by a ball and stick. 1, 2, and 3 represent the displacement of methyl carbon atoms in the 1800-psec snapshot structure measured from the initial structure. Black arrow represents the direction of each amino acid residue’s movement, and red arrow represents the rotational direction of TM2.

 
The recognition of HtrII by sRII
It is of great interest to clarify the reason why sRII specifically recognizes HtrII. To investigate the binding mechanism, we first examined hydrogen bonds between HtrII and sRII using the data of the ground-state simulation (Table 2Go). The criteria for the hydrogen bond are the same as described in the measurement of the interhelical hydrogen bond from helixF. A total of eight hydrogen bonds were detected between HtrII and sRII. All of the amino acid residues participating in these hydrogen bonds are not conserved in the corresponding sequence of BR (Halobacterium salinarium). This is a marked difference from the interhelical hydrogen bonds around helixF. BR cannot bind to the HtrII, that is, the amino acid residues (Arg162, Thr189, Thr191, Tyr199) in Table 2Go are important for the binding of HtrII.


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  		Table 2. Hydrogen bond between HtrII and sRII in the ground state
 
Second, we measured the interaction energy (VDW and Coulomb interaction) between HtrII and each amino acid residue of helixF and helixG using the MM_GBSA method (Kollmann et al. 2000; Fig. 10AGo). It is found from this measurement that five amino acid residues (R152, K157, R162, R164, Y199) have strong interaction with HtrII. The interaction energy was modified by including a solvent-effect energy term in addition to VDW and coulomb terms (Fig. 10BGo). The amino acid residue that has the largest interaction energy with HtrII is Tyr199, and the major factor for the interaction between Tyr199 and HtrII is not the hydrophilic, but the hydrophobic interaction term. This result is consistent with the result of the isothermal titration calorimetry experiment (Hippler-Mreyen et al. 2003) that reported the essential role of aromatic side chains of Tyr199 for proper binding of HtrII and sRII. Furthermore, the hydrogen bonding of Tyr199 with Asn74 of the transducer was detected in the X-ray structure (Gordeliy et al. 2002). Sudo et al. (2003) reported that this Tyr199–Asn74 hydrogen bonding might not be the only cause for the sRII–HtrII binding. In our present study, the hydrogen bonding of Tyr199 with the main chain of Phe28 of the transducer was observed, and this hydrogen bonding was more stable than that with Asn74. That is, Tyr199 assists the hydrophilic interaction between sRII and HtrII in addition to the significant contribution in hydrophobic interaction. This would be the reason why the interaction between Tyr199 and Phe28 of the transducer is essential for the sRII–HtrII binding, as reported by Hippler-Mreyen et al. (2003).



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  		Figure 10. (A) van der Waals(VDW) and Coulomb(EEL) interaction energy between HtrII and each amino acid residue of helixF and helixG (sRII). The residues marked with a circle had the large interaction with HtrII. (B) van der Waals (VDW), Coulomb(EEL), and solvation (SOL) interaction energy between HtrII and each amino acid residue of helixF and helixG (sRII).

 
Third, we calculated the change of binding affinity between HtrII and sRII using the MM_GBSA method, when the amino acid residues that had not been conserved in the sequence of BR in helixF and helixG were mutated to the corresponding residues of BR. Table 3Go shows the change of binding affinity for nine mutations. This measurement clearly demonstrates that three amino acid residues are important for the binding. One is Arg162 that has a positive charge. When Arg162 is mutated to Val, the affinity change from the wild type is 3.99 kcal/mol. This means that the substitution of Arg162 for Val decreases the binding affinity between HtrII and sRII. Several positively charged residues are distributed at the cytoplasmic end of helix F. These charged residues are not present in BR (Royant et al. 2001). Furthermore, these positively charged residues interact with the negatively charged cytoplasmic domain of TM2 helix of HtrII, the sequence of which is conserved among various species (Seidai et al. 1995). These results suggest that Arg162 mainly contributes to the remarkable stability of the sRII–HtrII complex, and the stability is due to the electrostatic interaction. In addition to this residue, Thr189 and Tyr199 show prominent affinity changes by the mutation of T189P and/or Y199V, and these two residues are also essential for the recognition of HtrII by sRII. These two amino acid residues are expected to also be important for sRI-HtrI binding in Halobacterium halobium sRI, which binds only its cognate transducer HtrI. Our sequence analysis shows a sound similarity in binding due to Thr189 between sRII–HtrII and sRI–HtrI systems, whereas a keen contrast is seen for Tyr199. As for Thr189, this amino acid residue is conserved in the corresponding position in Halobacterium halobium sRI. The residues at the corresponding positions of proton acceptor Glu43 and Ser62 in Natronobacterium pharaonis HtrII are Asp and Asn in Halobacterium halobium HtrI, respectively. Both amino acid residues of Asp and Asn can act as proton acceptors from Thr, like Glu43 and Ser62 in Natronobacterium pharaonis HtrII. We assume that the hydrogen bonds related to Thr189 in the sRII–HtrII system are conserved in the sRI–HtrI system. On the other hand, as for Tyr199, the residue at the corresponding position of this residue in Halobacterium halobium sRI is Phe. Phe has no hydroxyl side chain and cannot act as hydrogen-bond donor. In the sRII–HtrII system, Tyr199 has three hydrogen bonds with HtrII, and the counterpart residues in HtrII are Phe28, Thr33, and Asn74 (in Table 2Go). The residues at the corresponding positions of these residues in Halobacterium halobium HtrI are Ala, Leu, and Ala, respectively. All of these three residues have no side chains that can act as a proton acceptor. Because the hydrophobic interaction also shows a large contribution for Tyr199-relating interactions in the sRII–HtrII system, the hydrophobic interaction may be much emphasized in the sRI–HtrI system. This is a prominent difference in binding patterns between the sRII–HtrII and sRI–HtrI system. Further experimental and computational studies are needed from the viewpoint of molecular recognition.


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  		Table 3. Affinity changes calculated with MM_GBSA method
 

   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
Construction of the initial structure of the sRII–HtrII complex
For the ground-state simulation, the initial structure was constructed on the basis of the X-ray crystal structure (PDB code 1H2S [PDB] ; Gordeliy et al. 2002). For the K-state simulation, the crystal structure (PDB code 1GUE [PDB] ; Edman et al. 2002) was used, but this PDB code did not contain a HtrII part. To construct the sRII–HtrII complex in K-state, the sRII structure in 1GUE was superimposed on the sRII–HtrII complex of the ground-state using modeling software InsightII, and sRII part was substituted for the complex. No crystal structure was obtained for the sRII–HtrII complex in M-state. Hence, the initial structure of M-state was constructed by changing the protonation state of the initial structure of K-state. In K-state structure, Asp75 is unprotonated and Schiff base is protonated. On the other hand, Asp75 is protonated and Schiff base is unprotonated in M-state structure.

Construction of computational models for the protein–lipid bilayer–water solvent system
To reproduce the environmental condition for the membrane protein, the model structure of a lipid bilayer was constructed. For the component of lipid bilayer, we selected phosphatidylglycerophosphate monomethyl ester (PGP-Me), which is a main component of purple membrane (PM) (Kates et al. 1993). This lipid is considered to be essential for the photocycle expression of the other retinal protein in Halobacterium salinarium, because the photocycle activity of retinal protein decreases without PGP-Me (Joshi et al. 1998). First, a piece of the lipid bilayer consisting of 160 PGP-Me molecules was constructed. Second, 56 PGP-Me molecules at the center area of the lipid bilayer were removed for the sake of making the cavity in which the sRII–HtrII complex was placed. Third, the sRII–HtrII complex was inserted into the cavity using modeling software (InsightII). A total of 104 lipid molecules were arranged around the sRII–HtrII complex. Finally, 9973 water molecules were generated at the upper and the lower sides of the sRII–HtrII–lipid bilayer complex up to 7 Å from the edge of lipid molecules (Fig. 1Go).

Computational details
The simulations and analyses described in this work were carried out using the AMBER program package (Case et al. 2002), together with the Amber parm99 force field (Wang et al. 2000, except for retinal. Atomic charges and stable conformation of the retinal Schiff base were deduced from ab initio quantum chemical calculations, where density functional theory (DFT) (Lee et al. 1988; Becke 1993) were applied on a whole retinal Schiff-base structure using GAUSSIAN98 (Frisch et al. 1998). The hybrid Becke3LYP method and 6-31G** basis set were used for the DFT calculations. The charges were computed by the RESP method (Cieplak et al. 1995). In both unprotonated and protonated states, the same force field was assigned for retinal by using the antechamber module in the AMBER program package, except for the force parameter for C15-N torsion. The torsional potential was set to 28.76 kcal/mol for the protonated, and 30.0 for the unprotonated state (Tajkhorshid et al. 2000). An integration time step of the simulation was chosen to be 1 fsec. A cutoff distance of 10 Å for nonbonded interactions and a dielectric constant of {varepsilon} = 1 were used. A periodic boundary condition was applied, and the pressure was kept constant for the whole system.

Minimization, equilibration, and simulation data acquisition
First, potential energy minimizations were performed on each intermediate, starting from the respective initial structure. The total step of the minimization is 10,000; the first 3000 steps were executed by the steepest decent method; and the last 7000 steps were executed by the conjugated gradient method. Next, MD simulations were performed from the respective energy-minimized structure. The whole systems were gradually increased by heating up to 310 K for 60 psec, and then kept at 310 K for the next 500 psec. The trajectories at 310 K for 500 psec were considered to be the most probable structure under the physiological conditions, and were collected for analysis.

The figure of the structure in this work was drawn by molecular visualizing of software RasMol (Sayle and Milner-White 1995) and VMD (Humphrey et al. 1996).

Alignments and calculation of the degree of sequence conservation
Amino acid sequences of bacterial retinal proteins in different species were retrieved from DB (GENES, SwissProt, TrEMBL, TrEMBL_new, PIR, PRF, PDBSTR) using BLASTP 2.2.6 (Altschul et al. 1997). We used the Natronobacterium pharaonis sRII sequence as a query, and selected a total of 16 bacterial retinal proteins whose homological score is over 70. Classification of the 16 bacterial retinal proteins is as follows: BR-6, HR-4, sRI-2, and sRII-4. ClustalW (Thompson et al. 1994) was used for the multiple alignments. From the data of multiple alignment, the amino acid residues of Natronobacterium pharaonis sRII were examined in terms of the conservativeness in sequence among 16 bacterial retinal proteins.


   Footnotes
 
1 Present address: Japan Biological Information Research Center (JBIRC) Time 24 Bldg. 10F 2-45 Aomi, Koto-ku, Tokyo 135-0064 Japan. Back


   Acknowledgments
 
We thank Prof. Naoki Kamo at Hokkaido University for reading the manuscript and giving many important suggestions. The calculations were carried out by the DRIA system at the Graduate School of Pharmaceutical Sciences, Chiba University, and at the Institute of Media and Information Technology, Chiba University. This work was partly supported by a Grant-in-Aid for Center of Excellence (COE) research from the Ministry of Education, Science, Sport, and Culture, Japan.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
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