Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

<p>Abstract</p> <p>Background</p> <p>EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information.</p> <p>Results</p> <p>Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1.</p> <p>Conclusion</p> <p>Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.</p

Crystallization and preliminary X-ray crystallographic study of the extracellular domain of the 4-1BB ligand, a member of the TNF family

The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method

Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library

Recombinant EstE1 protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was then crystallized at 290 K by the hanging-drop vapour-diffusion method

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-2

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p>from archaeon and the mesophilic brefeldin A esterase (BFAE) from (PDB code ). The regions encompassing EstE1 dimerization motifs and the sequence blocks showing the amino acids involved in the formations of the catalytic triad and oxyanion hole are presented. Identical and similar residues have a grey background. Symbols: ●, amino acids forming a catalytic triad; ○, amino acids involved in oxynion hole formation; □ and ▯, amino acid residues involved in hydrophobic and ionic interactions at 1JJI dimeric interface, respectively; ▯, amino acid residues involved in ionic interactions at 1JKM dimeric interface; ■ and ▲ amino acid residues involved in hydrophobic and ionic interactions at EstE1 dimeric interface, respectively. Amino acid sequence alignment was performed as described previously [5]

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-5

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p> and EstE1(▯), in 20 mM potassium phosphate buffer (pH 7.0) were incubated at 80°C for the indicated times. Residual activities were then determined by measuring the amount of -nitrophenol released by esterase-catalyzed hydrolysis. The activity of a non-incubated sample was defined as 100

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-1

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p>ck representation. Val274, Phe276, and Leu299 are involved in the hydrophobic interactions. Arg270-Asp291 and Lys177-Glu295 form salt bridges. (B) The centrosymmetric conformation of the interface between the two monomers, consisting of centric hydrophobic interactions (red circle) and salt bridges. (C) A detailed view of the salt bridges that support the dimeric conformation of EstE1. The side chain of Arg270on the loop between the α8 helix and the β8 strand forms a salt bridge with Asp291on the α9 helix. An additional salt bridge is formed between the side chains of Lys177on the β6 strand and Glu295on the α9 helix. (D) A detailed view of the hydrophobic interaction interface observed in the EstE1 dimer. The hydrophobic core residues (Leu299 on the α9 helix, and Phe276 and Val274 on the β8 strand) are indicated. (E) A detailed view of the interface observed in a current AFEST dimer model [19]. Dimeric interactions of AFEST are supported by hydrogen bonds between Tyr280 and Gln303, and by a weak hydrophobic interaction through Val278

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-6

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p>elical segments and β-strands are shown in blue and yellow, respectively. G2 and G3 represent 3-helices. Helix α1 is not shown because of its disordered electron map. The catalytic triad containing residues Ser154, Asp251, and His281, are shown in stick representation. N and C denote the N and C termini, respectively