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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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Heme binding to HutZ. (A) Docking result of HutZ dimer-heme complex. HutZ dimer is shown in surface charge representation, with positive charge in blue, and negative charge in red. Heme molecule is labeled in yellow stick, with iron atom in red sphere. His63 and Arg92 that coordinate with heme iron atom are represented in magenta stick. The distance (Å) between His63/Arg92 and iron atom is in black dash. (B) Absorbance spectra of HutZ and its mutants reconstituted with heme. DM: double mutant.
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Figure 3: Heme binding to HutZ. (A) Docking result of HutZ dimer-heme complex. HutZ dimer is shown in surface charge representation, with positive charge in blue, and negative charge in red. Heme molecule is labeled in yellow stick, with iron atom in red sphere. His63 and Arg92 that coordinate with heme iron atom are represented in magenta stick. The distance (Å) between His63/Arg92 and iron atom is in black dash. (B) Absorbance spectra of HutZ and its mutants reconstituted with heme. DM: double mutant.

Mentions: A number of approaches were attempted to produce crystals of HutZ-heme complex, but no crystals were obtained. In this case, we carried out molecular docking to investigate the interactions between heme and HutZ. There are two large clefts in the HutZ dimer interfaces that are postulated, due to similarities with HugZ, to be the heme-binding pocket. The first-ranked resulting model of HutZ dimer-heme complex showed that the main contributors to the heme coordination included His63 loaded at the helix α2 of one monomer and Arg92 originating from strand β5 of the other (Figure 3A). These two residues cooperatively coordinated the iron atom in the center of the heme molecule, with the distance between His63 and the iron atom at approximately 3.6 Å and between Arg92 and the iron atom at 3.9 Å. UV absorption spectral analysis on native and mutant forms of HutZ-heme complexes were performed to explore whether His63 and Arg92 were both necessary for heme-binding. The Soret peak for the native HutZ-heme complex was at 410 nm; for the single mutant H63A reconstituted with heme, it was slightly shifted to 420 nm, indicating that His63 slightly affected heme binding. For R92A, the Soret band was not altered in comparison with the native HutZ-heme complex (Figure 3B), suggesting that Arg92 did not affect heme binding. However, there was no characteristic Soret peak detected for the double point mutant H63A-R92A reconstituted with heme. The Soret peak data collected were from full length HutZ and truncated HutZ (data not shown for full length HutZ). These observations indicated that at least one of these residues, His63 and Arg92, must exist to effectively coordinate the iron atom in heme, and either of them is sufficient. Additionally, eleven residues from monomer A (Ser42, Tyr43, Pro45, Ser58, Ile60, Ala61, Arg62, Arg65, Leu127, Asp132 and Phe133) and three residues from monomer B (Phe88, Thr94 and Phe145) around the heme-binding cleft participated in stabilizing the heme. These residues were fully conserved or replaced with similar residues in comparison with the residues stabilizing the heme group in the HugZ-heme complex (Additional file 3).


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)

Heme binding to HutZ. (A) Docking result of HutZ dimer-heme complex. HutZ dimer is shown in surface charge representation, with positive charge in blue, and negative charge in red. Heme molecule is labeled in yellow stick, with iron atom in red sphere. His63 and Arg92 that coordinate with heme iron atom are represented in magenta stick. The distance (Å) between His63/Arg92 and iron atom is in black dash. (B) Absorbance spectra of HutZ and its mutants reconstituted with heme. DM: double mutant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472187&req=5

Figure 3: Heme binding to HutZ. (A) Docking result of HutZ dimer-heme complex. HutZ dimer is shown in surface charge representation, with positive charge in blue, and negative charge in red. Heme molecule is labeled in yellow stick, with iron atom in red sphere. His63 and Arg92 that coordinate with heme iron atom are represented in magenta stick. The distance (Å) between His63/Arg92 and iron atom is in black dash. (B) Absorbance spectra of HutZ and its mutants reconstituted with heme. DM: double mutant.
Mentions: A number of approaches were attempted to produce crystals of HutZ-heme complex, but no crystals were obtained. In this case, we carried out molecular docking to investigate the interactions between heme and HutZ. There are two large clefts in the HutZ dimer interfaces that are postulated, due to similarities with HugZ, to be the heme-binding pocket. The first-ranked resulting model of HutZ dimer-heme complex showed that the main contributors to the heme coordination included His63 loaded at the helix α2 of one monomer and Arg92 originating from strand β5 of the other (Figure 3A). These two residues cooperatively coordinated the iron atom in the center of the heme molecule, with the distance between His63 and the iron atom at approximately 3.6 Å and between Arg92 and the iron atom at 3.9 Å. UV absorption spectral analysis on native and mutant forms of HutZ-heme complexes were performed to explore whether His63 and Arg92 were both necessary for heme-binding. The Soret peak for the native HutZ-heme complex was at 410 nm; for the single mutant H63A reconstituted with heme, it was slightly shifted to 420 nm, indicating that His63 slightly affected heme binding. For R92A, the Soret band was not altered in comparison with the native HutZ-heme complex (Figure 3B), suggesting that Arg92 did not affect heme binding. However, there was no characteristic Soret peak detected for the double point mutant H63A-R92A reconstituted with heme. The Soret peak data collected were from full length HutZ and truncated HutZ (data not shown for full length HutZ). These observations indicated that at least one of these residues, His63 and Arg92, must exist to effectively coordinate the iron atom in heme, and either of them is sufficient. Additionally, eleven residues from monomer A (Ser42, Tyr43, Pro45, Ser58, Ile60, Ala61, Arg62, Arg65, Leu127, Asp132 and Phe133) and three residues from monomer B (Phe88, Thr94 and Phe145) around the heme-binding cleft participated in stabilizing the heme. These residues were fully conserved or replaced with similar residues in comparison with the residues stabilizing the heme group in the HugZ-heme complex (Additional file 3).

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