Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B(ßß)

Takanori Tanaka1,3, Naofumi Miwa2, Satoru Kawamura2, Hitoshi Sohma3, Katsutoshi Nitta1 and Norio Matsushima4,5

1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060–0810, 2 Department of Biology, Graduate School of Science, Osaka University, Machikane-yama, Toyonaka, Osaka 560–0043, 3 School of Medicine and 4 School of Health Sciences, Sapporo Medical University, Chuo-ku, Sapporo, Hokkaido 060–8556, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
P26olf from olfactory tissue of frog, which may be involved in olfactory transduction or adaptation, is a Ca2+-binding protein with 217 amino acids. The p26olf molecule contains two homologous parts consisting of the N-terminal half with amino acids 1–109 and the C-terminal half with amino acids 110–217. Each half resembles S100 protein with about 100 amino acids and contains two helix–loop–helix Ca2+-binding structural motifs known as EF-hands: a normal EF-hand at the C-terminus and a pseudo EF-hand at the N-terminus. Multiple alignment of the two S100-like domains of p26olf with 18 S100 proteins indicated that the C-terminal putative EF-hand of each domain contains a four-residue insertion when compared with the typical EF-hand motifs in the S100 protein, while the N-terminal EF-hand is homologous to its pseudo EF-hand. We constructed a three-dimensional model of the p26olf molecule based on results of the multiple alignment and NMR structures of dimeric S100B(ßß) in the Ca2+-free state. The predicted structure of the p26olf single polypeptide chain satisfactorily adopts a folding pattern remarkably similar to dimeric S100B(ßß). Each domain of p26olf consists of a unicornate-type four-helix bundle and they interact with each other in an antiparallel manner forming an X-type four-helix bundle between the two domains. The two S100-like domains of p26olf are linked by a loop with no steric hindrance, suggesting that this loop might play an important role in the function of p26olf. The circular dichroism spectral data support the predicted structure of p26olf and indicate that Ca2+-dependent conformational changes occur. Since the C-terminal putative EF-hand of each domain fully keeps the helix–loop–helix motif having a longer Ca2+-binding loop, regardless of the four-residue insertion, we propose that it is a new, novel EF-hand, although it is unclear whether this EF-hand binds Ca2+. P26olf is a new member of the S100 protein family.

Keywords: calcium-binding protein/EF-hand/molecular modeling/p26olf/S100B


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The olfactory transduction mechanism has been shown to be similar to the phototransduction mechanism (Torre et al., 1995Go). In photoreceptors, several Ca2+-binding proteins such as calmodulin (Hsu and Molday, 1993Go), S-modulin or recoverin (Dizhoor et al., 1991Go; Kawamura, 1993Go) and GCAP (Palczewski et al., 1994Go; Dizhoor et al., 1995Go) were implicated as modulators in light adaptation (Kawamura, 1995Go). An influx of Ca2+ into the olfactory receptor cell is essential for olfactory adaptation (Kurahashi and Shibuya, 1990Go). Therefore, it is highly possible that olfactory adaptation is also mediated by Ca2+-binding proteins.

Very recently a Ca2+-binding protein named p26olf, which may be involved in the olfactory transduction or adaptation, was isolated from frog (Rana catesbeiana) olfactory epithelium (Miwa et al., 1998Go). The determined sequence consists of 217 amino acid residues. The sequence analysis showed that p26olf has two homologous parts consisting of the N-terminal half with amino acids 1–109 and the C-terminal half with amino acids 110–217. The two halves show close similarity to each other (identity 52.3%, similarity 67.0%) and moreover each resembles the S100 protein family (Figure 1Go).



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Fig. 1. Amino acid sequence alignments between p26olf and S100 family proteins. The multiple alignment contains the N- and C-terminal halves of p26olf and 18 S100 proteins. The regions corresponding to EF-hands were also aligned with the consensus sequence of EF-hand proposed (Kretsinger et al., 1991Go; Nakayama and Kretsinger, 1994Go). The two types of consensus sequences are shown at the very top. The consensus sequence of the normal EF-hand consists of 29 residues in a helix–loop–helix conformation; the Ca2+, if bound, is coordinated by six residues, whose positions are approximated by the vertices of an octahedron. Five of these, X, Y, Z, –X and –Z, usually have oxygen-containing side chains; Asp (D), Asn (N), Ser (S), Thr (T), Glu (E) or Gln (Q). The oxygen at position 16 (#), the –Y vertex, comes from the main chain and can be supplied by any amino acid. n refers to hydrophobic residues: Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) or Trp (W), the inner aspects of the {alpha}-helices. Gly (G) at position 15 permits a sharp bend ({Phi} = 90°, {Psi} = 0°) in the Ca2+-binding loop. Ile (I), Leu (L) or Val (V) at position 17 attaches the loop to the hydrophobic core of the molecule. The consensus sequence of the pseudo EF-hand consists of 31 residues (Kretsinger et al., 1991Go). Instead of coordinating Ca2+ with five side chains and one carbonyl oxygen at the vertex –Y (#), the pseudo EF-hand employs for carbonyl oxygen atoms `=O' and only one side chain, Glu at the –Z vertex. Loop positions are numbered from the –Y residue with carbonyl oxygens at 0, –3, –5 and –8. The first 15 residues of human S100D are MPAAWILWAHSHSEL and the final nine residues of human calgranulin B are KGPLGEGTP. The C-terminal domain (amino acids 101–1898) of human trichohyalin contains some types of tandem repeats. (N) and (C) in parentheses of p26olf indicate its N- and C-terminal halves, respectively. The prefixes are as follows: f, frog; r, rat; h, human; rt, rabbit; b, bovine; and p, porcine.

 
The S100 family is a group of abundant low molecular weight (10–12 kDa) acidic proteins with 79–110 amino acid residues that are highly enriched in nervous tissue. The S100 proteins have two helix–loop–helix Ca2+-binding structural motifs known as EF-hands (Figure 1Go). One of them, at the C-terminus, is a typical, normal EF-hand, similar to the EF-hands found in calmodulin and troponin C. Its consensus sequence is represented by 29 residues (Kretsinger et al., 1991Go; Nakayama and Kretsinger, 1994Go). The other at the C-terminus is referred to as a pseudo EF-hand with a consensus sequence consisting of 31 residues. This pseudo EF-hand has a lower affinity for Ca2+. The structure of calbindin D9k that was solved in the first place is in monomeric form (Szebenyi and Moffatt, 1986Go; Akke et al., 1992Go; Svensson et al., 1992Go; Kordel et al., 1993Go; Skelton et al., 1995Go). However, recent high-resolution nuclear magnetic resonance (NMR) and X-ray studies have demonstrated that calcyclin and S100B(ßß) exist in solution as the respective homodimers (Potts et al., 1995Go, 1996Go; Drohat et al., 1996Go, 1998Go; Kilby et al., 1996Go; Matsumura et al., 1998Go; Sastry et al, 1998Go; Smith and Shaw, 1998Go).

In order to investigate whether the single polypeptide chain of p26olf adopts a folding pattern similar to dimeric S100B or calcyclin, we performed a molecular modeling of p26olf. This will hopefully provide useful information to further our understanding of the principles of the three-dimensional structure of proteins. First, we carried out multiple alignment of p26olf with the S100 family proteins in detail. Second, we carried out the molecular modeling of p26olf based on the structures of the dimeric S100B in the Ca2+-free state. The predicted structure indicates that the single polypeptide chain of p26olf satisfactorily adopts a folding pattern remarkably similar to dimeric S100B. A loop linking the two S100-like domains, called a linker region, is fully accommodated. From the results of the sequence alignment, molecular modeling and circular dichroism (CD) measurements, we propose that the putative EF-hand located at the C-terminus in each S100-like domain of p26olf is a new type of EF-hand.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequence alignment

As the first step for the molecular modeling of p26olf, we performed multiple alignment of the two homologous halves of p26olf with 18 S100 family proteins. All amino-acid sequences examined, except p26olf, were obtained from SWISS-PROT and PIR protein sequence databases. The CLUSTALW program was used for multiple alignment (Thompson et al., 1994Go). During the process, the gap opening penalty and gap extension penalty were set at 10 and 0.05, respectively, and a BLOSUM matrix was used for the protein weight matrix. Simultaneously, the consensus sequences of the normal EF-hand and the pseudo EF-hand were also aligned to correspond with the EF-hand motifs in the two S100-like domains of p26olf and the S100 proteins (Kretsinger et al., 1991Go; Nakayama and Kretsinger, 1994Go).

Furthermore, we visually rearranged the whole single molecule of p26olf with 217 amino acids and a virtual polypeptide with 182 amino acids that is made by a peptide bond between the N-terminus of one subunit of S100ß and the C-terminus of the other subunit. The rearrangement was based on the results of the above-mentioned multiple alignment, with the exception of shifting the point of the gap insertion around Glu67 of S100ß to increase the local alignment score. This result was used for a homology modeling of p26olf.

Model building

We built models of p26olf on the basis of the results of the sequence alignment and the structures of dimeric S100B(ßß). In practice, we used two template proteins, rat and bovine S100B in the Ca2+-free state, since there are some differences concerning the interhelical angles for helix III/IV in these S100B structures (Drohat et al., 1996Go; Kilby et al., 1996Go).

Molecular modeling was performed on a Silicon Graphics INDIGO2 workstation using SYBYL 6.3 (Tripos, 1996Go). To generate a backbone framework, the coordinate data files for rat and bovine S100B were obtained from the Brookhaven Protein Data Bank (PDB) as 1SYM and 1CFP, respectively. A set of initial models of p26olf were constructed by mutation of the side chains in the dimeric S100B structures and modification of the four insertions found by the present alignment (this will be explained later). To model the p26olf single polypeptide chain, two molecules of the S100B dimer were joined. The first eight residues (Met1–Asn8) in the N-terminus and the last four residues (Leu214–Val217) in the C-terminus were omitted from the model structuring owing to a lack of applicable structural constraints for those regions. The inserted loops were roughly accommodated by using a loop conformational search program (SYBYL/Biopolymer). The loop search in two insertions of four residues (this will be explained later) was carried out with great care. The four-residue insertions are located on the loop region of the EF-hand adopting a helix–loop–helix structural motif. In addition to the four residues in the insertions, four more residues at the N-terminal side of the insertion were incorporated in the window region, because they constitute the loop of the EF-hand in the template structure. Major steric interferences caused by side chains were removed automatically and manually after visual inspection, such that the {chi}-angles of the side chains were kept to those of the template proteins as much as possible.

Refinement of the structures by energy minimization and molecular dynamics was performed according to the following protocol. All hydrogen atoms were included in the calculations and internal constraint for torsion angles ({omega}) at peptide bonds was imposed through the refinement. First, unfavorable van der Waals interactions of the residues belonging to only the inserted loops were optimized by 200 steps of energy minimization in an in vacuo situation using a Kollman all-atom force field implemented in SYBYL 6.3. Second, the entire protein was minimized for 1000 steps and molecular dynamics simulation was carried out at 300 K for 10 ps on only the linker region and again the energy was minimized for 1000 steps. These simulations were calculated in an in vacuo situation with a distance-dependent dielectric function with AMBER 4.1 (Pearlman et al., 1994). Third, an 8 Å layer of water molecules was added around the p26olf molecule. From this stage onwards we used a value of 1.0 for the dielectric constant with AMBER 4.1. The entire system consisting of p26olf and water molecules was minimized for 2000 steps. Then dynamics at 300 K was performed for 5 ps on the water molecules only, for 10 ps on the water molecules and the side chains and for 10 ps on the entire system, applying internal constraints for backbone dihedral angles ({Phi},{Psi}) in the helices. Lastly, after 20 ps of free dynamics, the protein was re-minimized for 30 000 steps.

Evaluation of models

Four possible models were finally constructed as candidates. The PROCHECK program was used to assess the stereochemical quality of the models (Laskowski et al., 1993Go). The secondary structure of every residue in the models was defined by the VADAR program (Wishart et al., 1994Go). Some additional evaluations were also made by examining the structures on a graphic display. Representative models were selected and submitted to further structural analyses and comparison with S100B. The comparison was performed by calculating pairwise r.m.s.d.s and representing the superposition using MOLMOL (Koradi et al., 1996Go).

Electrostatic potential calculations and representations

The electrostatic surface potential was calculated and displayed with the GRASP program (Nicholls et al., 1991Go). The linear Poisson–Boltzmann equations were solved using Amber fractional charges on each protein atom. The dielectric constants were set to 2 inside the protein and to 80 outside.

Circular dichroism (CD) measurements

The recombinant p26olf was expressed, purified and characterized. The method of sample preparation is given elsewhere (Miwa et al., 1998Go). Measurement of the CD spectrum of p26olf protein was performed with a JASCO J-725 spectropolarimeter with a thermostated holder. The pathlength was 0.1 cm. P26olf was dialyzed against 50 mM potassium phosphate (pH 7.4) before assay. Two solutions of p26olf (0.293 mg/ml) containing calcium-free buffer (0.2 mM EGTA) and calcium-saturated buffer (1.0 mM CaCl2) were placed in the cuvette and then the CD spectrum was acquired at a scan rate of 10 nm/min and the average of 16 scans was recorded. The mean residue ellipticity ([{theta}]) was calculated using a molecular weight of 24 000 Da. Estimation of the {alpha}-helical content was performed by using the method of Chen et al. (1972).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequence alignment

The multiple alignment demonstrated that the N- and C-terminal halves of p26olf particularly show higher identity and similarity with S100ß than with other S100 proteins except for S100{alpha} (Figure 1Go). Among S100ß, calcyclin and calbindin D9k, whose structures are now available (Potts et al., 1995Go, 1996Go; Drohat et al., 1996Go; Kilby et al., 1996Go; Szebenyi and Moffatt, 1986Go), S100ß shows the highest homology with the two halves. However, it appears that the degrees of homology are relatively low; the N-terminal half shows 28–29% identity and 50% similarity to S100ß and the C-terminal half shows 25% identity and 47% similarity. We used the structures of S100B(ßß) as a template for structural modeling of the p26olf single polypeptide chain, as explained in Materials and methods.

The next meaningful result was that both the N- and the C-terminal halves of p26olf have an obvious insertion of four residues, which definitely differs from all of the S100 proteins (Figure 1Go). Surprisingly, each of the two four-residue insertions is located in the region corresponding to the Ca2+-binding loop in a normal EF-hand motif of the S100 proteins, while the sequences corresponding to the pseudo EF-hand motif resulted in the alignments without any inserted gaps. Consequently, p26olf contains two pseudo EF-hands (EF-1 and EF-3) and two putative EF-hands (EF-2 and EF-4), as illustrated in Figure 2AGo. The other interesting result was that position 11 between X and Y positions in the consensus sequence of the normal EF-hand is occupied by Gly in the sequence of p26olf. They are Gly72 and Gly180 (Figure 1Go). Glycine does not occupy this position in the normal EF-hand of other proteins such as calmodulin and troponin C, so far as we know.



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Fig. 2. Schematic representation of the amino acid sequences of p26olf (A) and the alignments of p26olf with 217 amino acids and a virtual polypeptide with 182 amino acids that comprises S100ß dimer (B). (A) The p26olf sequence is divided into three parts; the N-terminal S100-like domain (N-domain) with amino acids 1–105, a linker region with amino acids 106–118, the C-terminal S100-like domain (C-domain) with amino acids 119–217. Each of the two S100-like domains contains a pseudo EF-hand (EF-1/EF-3) and a putative EF-hand (EF-2/EF-4). (B) A virtual polypeptide of S100ß with 182 amino acids is made from the peptide bond between the C-terminus of one subunit of S100ß dimer and its N-terminus of the other subunit. The regions corresponding to EF-hands (EF-1, EF-2, EF-3 and EF-4), linker region and two hinge regions are indicated above the alignments. Identical residues are indicated by black boxes and similar residues by gray boxes. Gaps inserted to maximize the alignment score are marked with dashes.

 
The p26olf sequence may be divided into three parts, the N-terminal S100-like domain (N-domain) with Met1–Cys105, a linker region with Pro106–Pro118 and the C-terminal S100-like domain (C-domain) with Thr119–Val217 (Figure 2AGo). From the alignment shown in Figure 2BGo, p26olf was found to have insertions at four points. The first insertion of one residue, Pro59, is located at a loop between EF-hands (called the hinge region) in the N-domain. Among the S100 proteins, both the length and amino-acid composition of the hinge regions are different from one another. The third and longest insertion with 13 residues (Pro106–Pro118) corresponds to the linker region connecting the N- and C-domains. It is rich in proline (four out of 13 amino acids) and poor in hydrophobic residues. Secondary structure predictions (Chou and Fasman, 1978Go; Garnier et al., 1978Go) for this region did not show any special characteristics. The second and fourth are insertions of the four residues which range from Lys77 to Gly80 in the N-domain and from Lys185 to Lys188 in the C-domain, respectively, as demonstrated from the above-mentioned multiple alignment (Figure 2BGo). In addition to these four insertions, there are N- and C-terminal extensions (Met1–Pro9 and Leu214–Val217). Since any conformational constraints for the two additional extensions cannot be defined, they were omitted from the model building, as noted in Materials and methods.

Possible structural models and evaluation

Four structures were finally constructed as possible models. The differences among these models mainly arose from those between the two template structures and in conformations of the 13-residue insertion constituting the linker region. In the template structures of rat and bovine S100B, the C{alpha}–C{alpha} distances between Met0 at the N-terminus of one subunit and Glu91 at the C-terminus of the other is 20.4 and 22.2 Å, respectively. It appears that the distance is enough to allow the 13-residue insertion of the linker region. Indeed, the N- and C-domains of p26olf were connected with no serious conformational change in any of the four models. Two stretches of four residues (Lys77–Gly80 and Lys185–Lys188) inserted in EF-2 and EF-4 were completely allowed to exist without steric conflict in the arrangement of helices. A proline residue inserted in the hinge region connecting the EF-hand pair EF-1 and EF-2 was also allowed to exist without steric conflict.

An analysis of the four predicted structures using a PROCHECK program indicated that the majority of residues occupied most favored or allowed regions in {Phi}{Psi} space: in each structure, >95% of all residues excluding proline and glycine fell in their regions. The {omega} angles were all trans-planar with a mean value near 180° and a standard deviation of about 6°. No bad contact between the residues was observed and the C{alpha} tetrahedral distortions were low. {chi}1 versus {chi}2 plots indicated that the side chain torsion angles occupied the normally observed range. Additionally, visual inspection of the models on a graphic display indicated that all of strong hydrophilic residues containing glutamic acid, aspartic acid, arginine and lysine were not oriented toward the protein core at all and so were accessible to the solvent.

Two representative structures were chosen from these four models; Model 1 and Model 2 originate from 1SYM and 1CFP, respectively. The two models have no characteristic conformation in the proline-rich linker region, as expected from the secondary structure predictions. Figure 3Go shows a Ramachandran plot of backbone dihedral angles ({Phi},{Psi}) of their models. In Model 1/2 only three/four residues had {Phi}{Psi} angles in disallowed regions and six/six residues had {Phi}{Psi} angles in generously allowed regions. The remaining {Phi}{Psi} angles of non-glycine and non-proline residues were present in most favored regions (77.5/75.3%) and additional allowed regions (17.6/19.2%). All of {Phi}{Psi} angles of proline and glycine residues were also in appropriate regions.




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Fig. 3. Ramachandran plots of the two predicted structures of p26olf. Model 1 (A) and Model 2 (B) were constructed from the template structures of rat and bovine S100B, respectively. 77.5/75.3% of the non-proline and non-glycine residues fall in the most favored regions (A, B and L), 17.6/19.2% are in the additional allowed regions (a, b, l and p), 3.3/3.3% fall in the generously allowed regions (~a, ~b, ~l and ~p) and 1.6/2.2% are in the disallowed region. Glycine residues are shown as triangles.

 
The secondary structure of every residue in the models was defined (Figure 4Go). Model 1 has eight helices (I, II, III, IV, V, VI, VII and VIII) and additionally one short helix ({alpha}-1), while Model 2 has additionally three short helices ({alpha}-1, {alpha}-2 and {alpha}-3) besides their eight helices. Helices {alpha}-1 and {alpha}-2 in Model 2 are seen in the template structure of bovine S100B (Kilby et al., 1996Go).



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Fig. 4. The secondary structure of the two predicted structures of p26olf. The two structures are Model 1 (from rat S100B) and Model 2 (from bovine S100B). Each of helices is sequentially labeled I–VIII. The secondary structure was defined by the VADAR program (Wishart et al., 1994Go). {square}, helix; , strand; ––-, coil.

 
There are significant differences between two template structures (rat and bovine S100B) which have an r.m.s.d. of 5.06 Å, as noted already. The two representative models were constructed from their S100B. The estimation containing the Ramachandran plot and secondary structure indicates that the two models are almost equal level.

P26olf model: comparison with the template (S100B)

The overall structure of p26olf single polypeptide chain having two S100-like domains is remarkably similar to the dimer structure of S100B (Figures 5 and 6GoGo). Each of the pairs of helices I + II, III + IV, V + VI and VII + VIII, that is linked by a loop, forms a helix–loop–helix structure which is characteristic of EF-hand (EF-1, EF-2, EF-3 and EF-4, respectively). Two putative EF-hands (EF-2 and EF-4) and two pseudo EF-hands (EF-1 and EF-3) will be compared later with those of S100B. EF-1 and EF-2 are linked by a loop between helices II and III, known as the hinge region. Also the hinge region (the loop between helices VI and VII) links EF-3 and EF-4. In Model 2 the hinge regions contain single turns of helix ({alpha}-1 and {alpha}-2). The EF-hand pairs interact with Ca2+-binding loops having a short antiparallel ß-strand or a short extended conformation at the end of each loop. The structure of each of the N- and C-domains of p26olf, consisting of two tightly packed EF-hands, belongs to the class of a unicornate-type four-helical bundle (Harris et al., 1994Go; Drohat et al., 1996Go), where helix I in the N-domain and helix V in the C-domain have a large angle with the three other, nearly parallel, helices in each bundle. The N- and C-domains are brought into close proximity by the antiparallel alignment of helices I and V and the antiparallel alignment of helices IV and VIII and the perpendicular association of the two pairs of antiparallel helices forms an X-type four-helical bundle, creating a feature similar to that seen in the dimer interface of Ca2+-free S100B (Drohat et al., 1996Go).



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Fig. 5. Ribbon diagrams of the predicted structure of p26olf related by a 90° rotation. (A) Model 1 (from rat S100B); (B) Model 2 (from bovine S100B). The N-terminal S100-like domain and the C-terminal S100-like domain are colored red and green, respectively, and the linker region white. This figure was made with MOLSCRIPT (Kraulis, 1991Go) and RASTER3D (Bacon and Anderson, 1988Go; Merritt and Murphy, 1994Go).

 


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Fig. 6. Stereo diagrams of C{alpha} superposition between the predicted structure of p26olf and NMR structure of S100B(ßß) in the Ca2+-free state. (A) Model 1 (from rat S100B); (B) Model 2 (from bovine S100B). The superposition was done using the corresponding template structure and optimized by minimization of the r.m.s.d. The four inserted regions and two extensions at the N- and C-termini of p26olf were excluded for calculating the r.m.s.d. The p26olf is illustrated by using red (the N-terminal S100-like domain), green (the C-terminal S100-like domain) and white (the linker region) and S100B dimer is drawn in blue. This figure was made with MOLMOL (Koradi et al., 1996Go).

 
A superposition of the C{alpha} trace of the whole molecule of p26olf with dimeric S100B is shown in Figure 6Go. Helices I, IV, V and VIII in the two p26olf models that constitute the X-type four-helix bundle are well conserved. In particular, helices IV and VIII strongly keep their positions in the template structures. In contrast, the lengths of helices II, III, VI and VII increase or decrease in comparison with those of the template structures (Figure 4Go). The global r.m.s.d. between p26olf and S100B structures is 2.96/3.68 Å, under the condition that the inserted regions and two extensions at the N- and C-terminus were excluded (Table IGo). The parts with greatest r.m.s.d. are in the loop region (Gly73 or Val74 to Glu81 and Thr182 or Ser181 to Met189) of EF-2 and EF-4, as expected. Moreover, in Model 2 two regions show larger r.m.s.d.s; one is Glu42 to Asp58 that are located on the C-terminus of helix II and helix {alpha}-1 and the other is Pro168 to Arg172 constituting a part of helix VII.


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Table I. Pairwise r.m.s.d.s between p26olf single polypeptide chain and dimeric S100B and between the part of EF-hands of p26olf and S100B
 
The C{alpha} superposition of the parts of EF-hands of p26olf and those of S100B are shown in Figure 7Go. Two pseudo EF-hands (EF-1 and EF-3) are very similar to those of S100B and two putative EF-hands (EF-2 and EF-4) satisfactorily keep the helix–loop–helix structural motif, regardless of the four-residue insertions in the region corresponding to the Ca2+-binding loop of a normal EF-hand, as noted already. However, the r.m.s.d.s are obviously larger than that between EF-1/EF-3 in p26olf and a pseudo EF-hand of S100B (Table IGo) and thus there seem to be some features in these two putative EF-hands. In Model 1 the insertions in EF-2 and EF-4 result in a kink of helices III and VII at their C-terminus and a bulging of the loop of their EF-hands. In Model 2 the insertion in EF-2 leads to an increase of length of helix III having a kink at its C-termini, whereas that in EF-4 leads to a decrease in length of helix VII and the appearance of a short helix ({alpha}-3). These may also be influenced by some small residues including Gly72 and Gly180 which are located on the loop region of the normal EF-hand.



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Fig. 7. Comparison of EF-hands between p26olf and Ca2+-free S100B by the C{alpha} superposition. (A) In Model 1 (from rat S100B); (B) in Model 2 (from bovine S100B). The comparison was optimized by minimization of the r.m.s.d. The left pictures of (A) and (B) indicate two pseudo EF-hands in p26olf; EF-1 with residues Lys18–Glu48 (drawn in red) and EF-3 with Ser127–Glu157 (green), are superimposed with the pseudo EF-hand with Ala9–Glu39 in S100B (blue). The right pictures of (A) and (B) indicate two putative EF-hands in p26olf; EF-2 with residues Ile62–Met94 (red) and EF-4 with Ile170–Ile202 (green), are similarly superimposed with the normal EF-hand with analogous residues Val52–Val80 in S100B (blue).

 
Hydrophobic core

The main feature of two models of p26olf is the existence of an extensive hydrophobic core running through the whole molecule, similar to that seen in dimeric S100B (Drohat et al., 1996Go). The hydrophobic core extends from the X-type four-helix bundle at the two domains' interface into the unicornate-type four-helix bundle of each domain. In Model 1/2, 59/62 out of 65 hydrophobic residues consisting of Leu, Ile, Val, Met, Phe and Tyr contribute to the formation of the hydrophobic core. Most of these hydrophobic residues are conserved or conservatively substituted in S100 proteins. The hydrophobic residues exposed to solvent are Ile62, Val74, Met103, Val113, Met189 and Val211 in Model 1 and Ile62, Met189 and Val211 in Model 2.

The hydrophobic residues at the domain interface of the two models include Met12, Ile19, Ile20, Phe23, Tyr26, Phe44, Met45, Leu49, Phe52, Phe84, Phe87, Ile91, Leu102, Met121, Ile128, Val129, Tyr132, Tyr135, Phe153, Met154, Leu158, Phe161, Phe192, Phe195, Ile199 and Phe210, which correspond to the conserved residues at the dimer interface of S100B (Drohat et al., 1996Go).

Moreover, some non-conserved residues including Phe38 and Tyr147 are found to participate in the hydrophobic interactions in the two models. The strongly hydrophobic residue Phe38, that is occupied by the hydrophilic residue Lys in the consensus sequence of the pseudo EF-hand motif, exhibits a hydrophobic interaction with Leu82 in a loop between helices III and IV. A similar hydrophobic interaction is found between Tyr147 (corresponding to Lys in the pseudo EF-hand) and Val178 in the C-terminus of helix VII. Met95 and Val96 at the C-terminus of helix IV and Met203 and Val204 at the C-terminus of helix VIII, all of which correspond to Thr in S100ß, are involved in hydrophobic interactions of the domain interface. Met190 (corresponding to Cys in S100ß) and in Model 2 three residues (Val74, Met103 and Val113) participate in hydrophobic interactions.

Solution conformation of p26olf

Typical far-UV CD spectra of p26olf in the Ca2+-free state and the Ca2+-saturated state are shown in Figure 8Go. The CD spectrum of the Ca2+-free p26olf is similar to that of Ca2+-free S100B. In the Ca2+-free state, the rate of [{theta}]222 which was used for a simple estimation of {alpha}-helical content was nearly –16 070° cm2/dmol, whereas in the Ca2+-saturated state the rate significantly changed to –21550°cm2/dmol. The apparent {alpha}-helical content calculated from the CD data was 45% in the Ca2+-free state and 63% in the Ca2+-saturated state. A value of 45% for Ca2+-free p26olf is comparable to the predictions from Model 1 (51%) and Model 2 (56%).



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Fig. 8. Circular dichroism spectra of p26olf in the Ca2+-free state and in the Ca2+-saturated state. The peptide solutions in 0.2 mM EGTA and in 1.0 mM CaCl2, pH 7.4, were placed in quartz cell of 0.1 cm pathlength. [{theta}] is the mean residue ellipticity. Thick solid line, p26olf in the Ca2+-free state; dashed line, p26olf in the Ca2+-saturated state; thin solid line, bovine S100B in the the Ca2+-free state (Mani et al., 1982Go).

 
With p26olf, analysis of the CD data indicated an increase of 18% in apparent {alpha}-helical content by the addition of Ca2+ (Figure 8Go). In contrast, with S100B and S100A, the CD data showed a decrease in apparent {alpha}-helical content upon Ca2+ binding to these proteins (Mani et al., 1982Go, 1983Go). However, recent NMR and X-ray analyses demonstrated that the {alpha}-helical content in the Ca2+-free state was almost equal to that in the Ca2+-saturated state (Amburgey et al., 1995Go; Kilby et al., 1995Go) and revealed that a drastic conformational change occurs upon Ca2+ binding to S100B (Drohat et al., 1998Go; Matsumura et al., 1998Go; Smith and Shaw, 1998Go). At this stage we consider that the observed CD spectral change of p26olf at high Ca2+ concentration is due to conformational changes, being indicative of Ca2+-binding to p26olf.

Electrostatic potentials

S100 family proteins including S100B and S100A are usually acidic proteins having a number of negatively charged residues. In contrast, p26olf is a neutral protein with a pI of 6.9. Indeed, a total of 29 negatively charged residues consisting of glutamic acid and aspartic acid is almost equal to 28 positively charged residues consisting of arginine and lysine. It is interesting to compare the electrostatic potentials of p26olf and S100B. As shown in Figure 9Go, a broader part of the surface of p26olf have zero or close to zero for the electrostatic potential and a few positive or negative patches are present on the surface. On the other hand, S100B is surrounded by a strong negative electrostatic potential, especially in a cleft in the center of the molecule, as also noted in calcyclin (Potts et al., 1995Go; Kilby et al., 1996Go).



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Fig. 9. Electrostatic surface representation of the predicted structure of p26olf and Ca2+-free S100B determined by NMR. (A) Model 1 (from rat S100B); (B) Model 2 (from bovine S100B); (C) the structure of rat S100B in the Ca2+-free state (Drohat et al., 1996Go). Blue represents regions of positive potentials and red regions of negative potentials. The orientation of the molecule is the same as in the lower picture of Fig. 5Go. This figure was made with GRASP (Nicholls et al., 1992).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Relationship between p26olf single polypeptide chain and dimeric S100B: examples in other proteins

The present molecular modeling indicated that the overall structure of p26olf single polypeptide chain is remarkably similar to that of dimeric S100B (Figures 5 and 6GoGo). Their structures are characterized by a unicornate-type and an X-type four-helix bundle. A similar relationship is frequently observed among a variety of proteins adopting another type of four-helix bundle, e.g. between the RNA-binding protein Rop and myohenerythrin. The Rop protein molecule is a dimer in which the two subunits are arranged such that a bundle of four {alpha}-helices is formed, with their long axes aligned. A similar arrangement of four {alpha}-helices is formed within a single polypeptide chain of myohenerythrin. Such a single chain forming a four-helix bundle is also observed in a non-heme iron containing oxygen transport protein in marine worms, cytochrome c', cytochrome b562 (which are heme-containing electron carriers), ferritin (which is a storage molecule for Fe atoms in eukaryotic cells) and the coat protein of tobacco mosaic virus (Branden and Tooze, 1991Go). The four helices pack against each other forming a hydrophobic core, which is also similar to the X-type or unicornate-type four-helix bundle in p26olf and S100B.

It is also interesting to note the enzyme structures of 5-carboxymethyl-2-hydroxymuconate isomerase (CHMI) and 4-oxalocrotonate tautomerase (4-OT) that catalyze the isomerization of unsaturated ketones (Subramanya et al., 1996Go). The CHMI trimer and the 4-OT hexamer show similar structures to each other, in which the subunits in both molecules are arranged around a 3-fold axis with active sites on the outside of the molecule, but the two parts of each pair of 4-OT dimers interact with the other to form a similar conformation to CHMI monomer. A similar arrangement was also identified in the macrophage migration inhibitory factor (MIF), which exists as a trimer in the crystal (Suzuki et al., 1996Go). Interestingly, MIF, CHMI and 4-OT have no apparent sequence homology to each other.

Hence the implication is that the loops linking structural domains, which were frequently observed in the structures of the proteins, contribute less to or may not be necessary for the stabilization of their structures.

Putative EF-hand motif in p26olf

It is clear that the two putative EF-hands (EF-2 and EF-4) having the four-residue insertions are never identified as a normal or a pseudo EF-hand motif, according to Kretsinger's criterion (Kretsinger et al., 1991Go). However, the residues at all of the `n' and `I' positions which were occupied by hydrophobic residues in the consensus sequence were completely conserved (Figure 1Go). Indeed, the two putative EF-hands in the two models kept the helix–loop–helix structure in which the hydrophobic residues at the `n' and `I' positions participate in the formation of the hydrophobic core (Figures 5, 6 and 7GoGoGo), although the four-residue insertions induce the deformation of some helices and/or the creation of a short helix. Therefore, we propose that both EF-2 and EF-4 are a new, novel EF-hand having a longer Ca2+-binding loop than those of normal and pseudo EF-hand motifs, although it is unclear whether EF-2 and EF-4 really bind Ca2+.

Recently, a similar situation was observed in the structure of the C-terminal domain of human BM-40 (SPARC, osteonectin) containing an EF-hand pair in which the two EF-hands interact canonically (Hohenester et al., 1996Go). However, in the first EF-hand a one-residue insertion is accommodated by a cis-peptide bond and by substituting a carboxylate by a peptide carbonyl as a Ca2+-ligand and the EF-hand actually binds Ca2+.

Binding of Ca2+ to p26olf

The CD spectra indicate that a conformational change occurs upon binding Ca2+ to p26olf (Figure 8Go). The structures of S100B in both the Ca2+-free state and the Ca2+-saturated state are available (Drohat et al., 1996Go, 1998Go; Kilby et al., 1996Go; Matsumura et al., 1998Go; Smith and Shaw, 1998Go). These structures reveal that the binding of Ca2+ to S100B leads to large changes in the conformation. Interestingly, the change occurs not in the pseudo EF-hand but the normal EF-hand, which is mainly represented by a change in the position of helix III relative to other helices. Furthermore, among the structures of S100B, calcyclin and calbindin D9k in the Ca2+-free state the biggest difference is in the location of helix III (Kilby et al., 1996Go). The pairwise r.m.s.d.s between eight helices of p26olf and the corresponding helices of S100B showed the largest value in helix III/VII forming the novel EF-hands (EF-2 and EF-4) of p26olf (not shown). Taken together, either or both of the two novel EF-hands in p26olf is presumed to be capable of the binding of Ca2+ and thus helix III/VII may be responsible for the Ca2+-dependent conformational change.

So far, Zn2+ binding has been demonstrated directly for S100B (Baudier et al., 1986Go), calcyclin (Pedrocchi et al., 1994Go), calgranulin (Dell'Angellica et al., 1994Go) and S100A3 (Fohr et al., 1995Go). Ca2+- and Zn2+-binding sites are different (Baudier and Gerard, 1983Go). Histidine residues are known to be ubiquitous ligands for Zn2+ ions in proteins. S100A3, with the highest cysteine content (10%), binds Zn2+ with high affinity (Figure 1Go). Cysteine and histidine of p26olf are conserved at the C-terminal region of each domain when compared with S100ß (Figure 1Go). It will be of significant importance to investigate whether p26olf binds Zn2+.

Implications on the function of p26olf

P26olf has been suggested to play a role in olfactory transduction, since p26olf is enriched in the distal segment of the cilia (Miwa et al., 1998Go). Also, p26olf may participate in olfactory adaptation because of the essential role of Ca2+ in the adaptation. We consider that p26olf could interact with some (unknown) proteins, since the S100 protein family interact with a variety of proteins such as fructose-1,6-biphosphate aldolase, annexin II, annexin VI and p53 (Donato, 1991Go; Schafer and Heizmann, 1996Go). The hinge region, which connects the EF-hands in an S100ß subunit, and the C-terminal loop are postulated to be involved in binding specific target proteins (Krebs et al., 1995Go; Kligman and Hilt, 1988Go). Indeed, a surface which is formed from the hinge region and the C-terminal loop on the opposite side of the S100ß subunit from the Ca2+-binding loop is ideal for interaction with target protein (Drohat et al., 1996Go). The linker region in the p26olf structure is close to the C-terminal loop of the S100ß in the S100B structure (Figure 5Go). In p26olf the linker region in addition to the hinge region might play an important role in interactions with target proteins.

Moreover, it is significant to note that p26olf is a neutral protein differing from acidic S100 proteins. This implies that p26olf may function in an environment different from S100.

Implications for the evolution of p26olf

Sequence analysis indicated that p26olf consists of two S100-like domains including the novel EF-hands with high homology to each other (Figure 1Go). Molecular modeling also indicates that the structure of each domain is remarkably similar. Thus it is inferred that the p26olf gene is made from a duplication of either domain. Recent X-ray studies demonstrated the presence of some types of novel EF-hands (Hohenester et al., 1996Go; Kretsinger, 1996Go, 1997Go; Blanchard et al., 1997Go; Lin et al., 1997Go). Kretsinger (1996) considers that a reconsideration of the evolutionary and functional relationship among the members of diverse EF-hand proteins is necessary.

Conclusion

The p26olf single polypeptide chain having two S100-like domains adopts a folding pattern remarkably similar to dimeric S100B. Each domain consists of a unicornate-type four-helix bundle that interacts with the others in an antiparallel manner and so forms an X-type four-helix bundle between the two domains. The CD results support the predicted structure. We propose that a putative EF-hand in each domain of p26olf is a new, novel EF-hand motif. An experimental study on the ability of Ca2+-binding to the novel EF-hands proposed here is in progress.

The atomic coordinates of the models are available from the authors and will be submitted to the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973.


    Acknowledgments
 
We are grateful to Professor Hideo Kanoh (Sapporo Medical University) and Professor Michio Yazawa (Hokkaido University) for their helpful support, to Dr Toshio Ohyanagi (Sapporo Medical University) for assistance with the AMBER program and to Kengo Kinosita (Kyoto University) for providing useful references. This work was supported in part by the Suhara Foundation (to N.Matsushima) and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to N.Matsushima).


    Notes
 
5 To whom correspondence should be addressed. E-mail: matusima{at}shs.sapmed.ac.jp Back


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Received October 9, 1999; revised January 22, 1998; accepted February 17, 1999.