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Crystal structure of HutZ, a heme storage protein from Vibrio cholerae: A structural mismatch observed in the region of high sequence conservation.

Liu X, Gong J, Wei T, Wang Z, Du Q, Zhu D, Huang Y, Xu S, Gu L - BMC Struct. Biol. (2012)

Bottom Line: This mismatch initiates more divergent structural characteristics towards their C-terminal regions, which are essential features for the heme-binding of HugZ as a heme oxygenase.HutZ's deficiency in heme oxygenase activity might derive from its residue shift relative to the heme oxygenase HugZ.This residue shift also emphasized a limitation of the traditional template selection criterion for homology modeling.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, 250100, China.

ABSTRACT

Background: HutZ is the sole heme storage protein identified in the pathogenic bacterium Vibrio cholerae and is required for optimal heme utilization. However, no heme oxygenase activity has been observed with this protein. Thus far, HutZ's structure and heme-binding mechanism are unknown.

Results: We report the first crystal structure of HutZ in a homodimer determined at 2.0 Å resolution. The HutZ structure adopted a typical split-barrel fold. Through a docking study and site-directed mutagenesis, a heme-binding model for the HutZ dimer is proposed. Very interestingly, structural superimposition of HutZ and its homologous protein HugZ, a heme oxygenase from Helicobacter pylori, exhibited a structural mismatch of one amino acid residue in β6 of HutZ, although residues involved in this region are highly conserved in both proteins. Derived homologous models of different single point variants with model evaluations suggested that Pro140 of HutZ, corresponding to Phe215 of HugZ, might have been the main contributor to the structural mismatch. This mismatch initiates more divergent structural characteristics towards their C-terminal regions, which are essential features for the heme-binding of HugZ as a heme oxygenase.

Conclusions: HutZ's deficiency in heme oxygenase activity might derive from its residue shift relative to the heme oxygenase HugZ. This residue shift also emphasized a limitation of the traditional template selection criterion for homology modeling.

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Related in: MedlinePlus

Sequence and structural comparisonsof HutZ and HugZstructural shift regions. (A) Sequence correspondence based on sequence alignment. Secondary structures are labeled above or below sequences. The shaded region represents the hypothetical β7 of HutZ, which does not exist in present crystal structure. (B) Sequence correspondence based on structure superimposition. Different from A, the four-residue-long corner of HutZ is aligned to three-residue-long corner of HugZ. Secondary structures are labeled above or below sequences. (C) Structural superimposition of HutZ and HugZ. Protein backbones are shown in trace representation with side chains in bond. (D) C-terminals of main chains of β6-2 and β9-2 point in different directions forming a 60° angle.
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Figure 4: Sequence and structural comparisonsof HutZ and HugZstructural shift regions. (A) Sequence correspondence based on sequence alignment. Secondary structures are labeled above or below sequences. The shaded region represents the hypothetical β7 of HutZ, which does not exist in present crystal structure. (B) Sequence correspondence based on structure superimposition. Different from A, the four-residue-long corner of HutZ is aligned to three-residue-long corner of HugZ. Secondary structures are labeled above or below sequences. (C) Structural superimposition of HutZ and HugZ. Protein backbones are shown in trace representation with side chains in bond. (D) C-terminals of main chains of β6-2 and β9-2 point in different directions forming a 60° angle.

Mentions: Structural superimposition revealed that the HutZ monomer shared high structural similarity with HugZ monomer (structure RMSD = 1.645 Å and sequence identity = 35%), which is a heme oxygenase from Helicobacter pylori[12,13]. Sequence alignment showed that the HugZ N-terminal domain (residues 1–80) was absent in HutZ, and the C-terminal loop (residues 238–249) of HugZ was variable in HutZ. In HutZ, the β6 contained a four-residue-long corner structure (Pro140-Gly143) that divides β6 into two segments (Figure 1B), designated as β6-1 (Phe133-Gln139) and β6-2 (Leu144-Gly148) respectively, while the corresponding β9 in HugZ is also divided into two segments β9-1 (Phe208-Asp214) and β9-2 (Gly218-Gly223) by a three-residue-long corner (Phe215-Glu217) (Figure 4A). The Fo_Fc omit map clearly revealed the positions of the four amino acid residues composing the corner of β6 in HutZ (Additional file 4). Very interestingly, although the segment Gly143-Tyr153 in HutZ was identical to the segment Gly218-Tyr228 in HugZ except for two pairs of amino acid residues (Arg219 of HutZ and Leu144 of HugZ, Phe225 of HutZ and Gly150 of HugZ) (Figure 4A), these residues did not match each other in one-to-one correspondence in the superimposition of three-dimensional structures (Figure 4B and C). Rather, β6-2 was shifted frontward by one amino acid residue relative to β9-2. For example, Phe145 of HutZ did not match Phe220 of HugZ, but corresponded to the preceding Arg219. This structural mismatch was substantially represented as changes in hydrogen bonding patterns between adjacent β-strands. Two adjacent β-strands form a hydrogen bond network in which the N-H groups or C = O groups of one strand establish hydrogen bonds with the C = O groups or N-H groups of another. Normally, there are two hydrogen bonds (N-H-O and O-H-N) on every second amino acid residue in one strand. In the HutZ and HugZ structures, the hydrogen bonding patterns were both regular on β6-1 and β9-1 (Additional file 5. A and B). However, in the four-residue-long corner of HutZ, no hydrogen bonds were detected, whereas in the three-residue-long corner of HugZ, two consecutive hydrogen bonds existed on Lys216 and Glu217. In this vein, after both corner regions, the correspondence between the hydrogen bond providing residues on β6-2 and β9-2 were not in line with that of their sequence alignment (Additional file 5. C). There was a mismatch of one amino acid residue.


Crystal structure of HutZ, a heme storage protein from Vibrio cholerae: A structural mismatch observed in the region of high sequence conservation.

Liu X, Gong J, Wei T, Wang Z, Du Q, Zhu D, Huang Y, Xu S, Gu L - BMC Struct. Biol. (2012)

Sequence and structural comparisonsof HutZ and HugZstructural shift regions. (A) Sequence correspondence based on sequence alignment. Secondary structures are labeled above or below sequences. The shaded region represents the hypothetical β7 of HutZ, which does not exist in present crystal structure. (B) Sequence correspondence based on structure superimposition. Different from A, the four-residue-long corner of HutZ is aligned to three-residue-long corner of HugZ. Secondary structures are labeled above or below sequences. (C) Structural superimposition of HutZ and HugZ. Protein backbones are shown in trace representation with side chains in bond. (D) C-terminals of main chains of β6-2 and β9-2 point in different directions forming a 60° angle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Sequence and structural comparisonsof HutZ and HugZstructural shift regions. (A) Sequence correspondence based on sequence alignment. Secondary structures are labeled above or below sequences. The shaded region represents the hypothetical β7 of HutZ, which does not exist in present crystal structure. (B) Sequence correspondence based on structure superimposition. Different from A, the four-residue-long corner of HutZ is aligned to three-residue-long corner of HugZ. Secondary structures are labeled above or below sequences. (C) Structural superimposition of HutZ and HugZ. Protein backbones are shown in trace representation with side chains in bond. (D) C-terminals of main chains of β6-2 and β9-2 point in different directions forming a 60° angle.
Mentions: Structural superimposition revealed that the HutZ monomer shared high structural similarity with HugZ monomer (structure RMSD = 1.645 Å and sequence identity = 35%), which is a heme oxygenase from Helicobacter pylori[12,13]. Sequence alignment showed that the HugZ N-terminal domain (residues 1–80) was absent in HutZ, and the C-terminal loop (residues 238–249) of HugZ was variable in HutZ. In HutZ, the β6 contained a four-residue-long corner structure (Pro140-Gly143) that divides β6 into two segments (Figure 1B), designated as β6-1 (Phe133-Gln139) and β6-2 (Leu144-Gly148) respectively, while the corresponding β9 in HugZ is also divided into two segments β9-1 (Phe208-Asp214) and β9-2 (Gly218-Gly223) by a three-residue-long corner (Phe215-Glu217) (Figure 4A). The Fo_Fc omit map clearly revealed the positions of the four amino acid residues composing the corner of β6 in HutZ (Additional file 4). Very interestingly, although the segment Gly143-Tyr153 in HutZ was identical to the segment Gly218-Tyr228 in HugZ except for two pairs of amino acid residues (Arg219 of HutZ and Leu144 of HugZ, Phe225 of HutZ and Gly150 of HugZ) (Figure 4A), these residues did not match each other in one-to-one correspondence in the superimposition of three-dimensional structures (Figure 4B and C). Rather, β6-2 was shifted frontward by one amino acid residue relative to β9-2. For example, Phe145 of HutZ did not match Phe220 of HugZ, but corresponded to the preceding Arg219. This structural mismatch was substantially represented as changes in hydrogen bonding patterns between adjacent β-strands. Two adjacent β-strands form a hydrogen bond network in which the N-H groups or C = O groups of one strand establish hydrogen bonds with the C = O groups or N-H groups of another. Normally, there are two hydrogen bonds (N-H-O and O-H-N) on every second amino acid residue in one strand. In the HutZ and HugZ structures, the hydrogen bonding patterns were both regular on β6-1 and β9-1 (Additional file 5. A and B). However, in the four-residue-long corner of HutZ, no hydrogen bonds were detected, whereas in the three-residue-long corner of HugZ, two consecutive hydrogen bonds existed on Lys216 and Glu217. In this vein, after both corner regions, the correspondence between the hydrogen bond providing residues on β6-2 and β9-2 were not in line with that of their sequence alignment (Additional file 5. C). There was a mismatch of one amino acid residue.

Bottom Line: This mismatch initiates more divergent structural characteristics towards their C-terminal regions, which are essential features for the heme-binding of HugZ as a heme oxygenase.HutZ's deficiency in heme oxygenase activity might derive from its residue shift relative to the heme oxygenase HugZ.This residue shift also emphasized a limitation of the traditional template selection criterion for homology modeling.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, 250100, China.

ABSTRACT

Background: HutZ is the sole heme storage protein identified in the pathogenic bacterium Vibrio cholerae and is required for optimal heme utilization. However, no heme oxygenase activity has been observed with this protein. Thus far, HutZ's structure and heme-binding mechanism are unknown.

Results: We report the first crystal structure of HutZ in a homodimer determined at 2.0 Å resolution. The HutZ structure adopted a typical split-barrel fold. Through a docking study and site-directed mutagenesis, a heme-binding model for the HutZ dimer is proposed. Very interestingly, structural superimposition of HutZ and its homologous protein HugZ, a heme oxygenase from Helicobacter pylori, exhibited a structural mismatch of one amino acid residue in β6 of HutZ, although residues involved in this region are highly conserved in both proteins. Derived homologous models of different single point variants with model evaluations suggested that Pro140 of HutZ, corresponding to Phe215 of HugZ, might have been the main contributor to the structural mismatch. This mismatch initiates more divergent structural characteristics towards their C-terminal regions, which are essential features for the heme-binding of HugZ as a heme oxygenase.

Conclusions: HutZ's deficiency in heme oxygenase activity might derive from its residue shift relative to the heme oxygenase HugZ. This residue shift also emphasized a limitation of the traditional template selection criterion for homology modeling.

Show MeSH
Related in: MedlinePlus