Hyongi Chon

Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3Å resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central αβα substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing.

The structural and biochemical characterization of human RNase H2 complex reveals the molecular basis for substrate recognition and Aicardi-Goutieres syndrome defects.

RNase H2 cleaves RNA sequences that are part of RNA/DNA hybrids or that are incorporated into DNA, thus, preventing genomic instability and the accumulation of aberrant nucleic acid, which in humans induces Aicardi-Goutières syndrome, a severe autoimmune disorder. The 3.1 Å crystal structure of human RNase H2 presented here allowed us to map the positions of all 29 mutations found in Aicardi-Goutières syndrome patients, several of which were not visible in the previously reported mouse RNase H2. We propose the possible effects of these mutations on the protein stability and function. Bacterial and eukaryotic RNases H2 differ in composition and substrate specificity. Bacterial RNases H2 are monomeric proteins and homologs of the eukaryotic RNases H2 catalytic subunit, which in addition possesses two accessory proteins. The eukaryotic RNase H2 heterotrimeric complex recognizes RNA/DNA hybrids and (5′)RNA-DNA(3′)/DNA junction hybrids as substrates with similar efficiency, whereas bacterial RNases H2 are highly specialized in the recognition of the (5′)RNA-DNA(3′) junction and very poorly cleave RNA/DNA hybrids in the presence of Mg2+ ions. Using the crystal structure of the Thermotoga maritima RNase H2-substrate complex, we modeled the human RNase H2-substrate complex and verified the model by mutational analysis. Our model indicates that the difference in substrate preference stems from the different position of the crucial tyrosine residue involved in substrate binding and recognition.

Structure of amyloid β fragments in aqueous environments

Conformational studies on amyloid β peptide (Aβ) in aqueous solution are complicated by its tendency to aggregate. In this study, we determined the atomic‐level structure of Aβ28−42 in an aqueous environment. We fused fragments of Aβ, residues 10–24 (Aβ10−24) or 28–42 (Aβ28−42), to three positions in the C‐terminal region of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk‐RNase HII). We then examined the structural properties in an aqueous environment. The host protein, Tk‐RNase HII, is highly stable and the C‐terminal region has relatively little interaction with other parts. CD spectroscopy and thermal denaturation experiments demonstrated that the guest amyloidogenic sequences did not affect the overall structure of the Tk‐RNase HII. Crystal structure analysis of Tk‐RNase HII1−197–Aβ28−42 revealed that Aβ28−42 forms a β conformation, whereas the original structure in Tk‐RNase HII1−213 was α helix, suggesting β‐structure formation of Aβ28−42 within full‐length Aβ in aqueous solution. Aβ28−42 enhanced aggregation of the host protein more strongly than Aβ10−24. These results and other reports suggest that after proteolytic cleavage, the C‐terminal region of Aβ adopts a β conformation in an aqueous environment and induces aggregation, and that the central region of Aβ plays a critical role in fibril formation. This study also indicates that this fusion technique is useful for obtaining structural information with atomic resolution for amyloidogenic peptides in aqueous environments.

Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HII.

Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi–Goutières syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His‐tagged Saccharomyces cerevisiae RNase H2 (Sc‐RNase H2*) were constructed. Sc‐G42S*, Sc‐L52R*, and Sc‐K46W* contain single mutations in Sc‐Rnh2Ap*, Sc‐Rnh2Bp*, and Sc‐Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc‐RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc‐G42S* was greatly reduced compared to that of the wild‐type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk‐G10S, Tk‐G10A, Tk‐G10L, and Tk‐G10P, were constructed. All mutant proteins were less stable than the wild‐type protein by 2.9–7.6 °C in Tm. A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable.

Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.

BackgroundThe unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).ResultsTo clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.ConclusionsThese results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

Conformational contagion in a protein: structural properties of a chameleon sequence

Certain sequences, known as chameleon sequences, take both α‐ and β‐conformations in natural proteins. We demonstrate that a wild chameleon sequence fused to the C‐terminal α‐helix or β‐sheet in foreign stable proteins from hyperthermophiles forms the same conformation as the host secondary structure. However, no secondary structural formation is observed when the sequence is attached to the outside of the secondary structure. These results indicate that this sequence inherently possesses an ability to make either α‐ or β‐conformation, depending on the sequentially neighboring secondary structure if little other nonlocal interaction occurs. Thus, chameleon sequences take on a satellite state through contagion by the power of a secondary structure. We propose this “conformational contagion” as a new nonlocal determinant factor in protein structure and misfolding related to protein conformational diseases. Proteins 2007.

Crystal structure of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3 at 2.20 Å resolution

Introduction. Kynurenine aminotransferase (KAT; EC. 2.6.1.7) is an enzyme that catalyzes the irreversible transamination of L-kynurenine (L-Kyn) to produce kynurenic acid (KA). L-Kyn is a major metabolite in the degradation pathway of tryptophan, and KA acts as an endogenous antagonist of all three ionotropic excitatory amino acid receptors in the central nervous system. Enormous attention has recently been paid to structural and functional studies of KAT, because alteration in the endogenous KA level has been suggested to cause a number of brain and neuron diseases. In mammals, two KAT isozymes, KAT-I and KAT-II, have been identified and characterized. KAT-I and KAT-II are also referred to glutamine transaminase K and -aminoadipate aminotransferase, respectively. The crystal structures of human KAT-I (hKAT-I), and its homologues, such as glutamine-phenylpyruvate aminotransferase and aspartate aminotransferase from Thermus thermophilus HB8, have been determined. In contrast, neither the crystal structures of KAT-II nor those of its functional homologues have been determined. The atomic coordinates of a KAT-II homologue from Thermotoga maritima MSB8 (Tm-PAT) have been deposited in the Protein Data Bank (PDB) with accession number 1vp4. However, it remains to be determined whether this protein exhibits the KAT activity. KAT-I and KAT-II have been reported to differ in specific activity, substrate specificity, and sensitivity to inhibitors. To understand the structural bases for these differences, it is necessary to determine the crystal structures of KAT-II or its functional homologues. It seems difficult to construct a three-dimensional model for KAT-II based on the KAT-I structures because of the poor amino-acid sequence identities between KAT-I and KAT-II (11% for human enzymes). We have recently overproduced, purified, and characterized a KAT-II homologue from Pyrococcus horikoshii OT3 (phKAT-II). Crystallization and preliminary X-ray diffraction analysis of this protein have also been completed. This protein shows an amino-acid sequence identity of 30% to hKAT-II and exhibits the KAT activity in a homodimeric form. In this study, we solved the crystal structure of phKAT-II in order to understand a structural basis for the enzymatic function of KAT-II.

Gene Cloning and Biochemical Characterizations of Thermostable Ribonuclease HIII from Bacillus stearothermophilus

The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst–RNase HIII) was purified and biochemically characterized. Bst–RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu–RNase HIII). The enzymatic properties of Bst–RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu–RNase HIII. However, Bst–RNase HIII was more stable than Bsu–RNase HIII, and the temperature (T1⁄2) at which the enzyme loses half of its activity upon incubation for 10 min was 55 °C for Bst–RNase HIII and 35 °C for Bsu–RNase HIII. The optimum temperature for Bst–RNase HIII activity was also shifted upward by roughly 20 °C as compared to that of Bsu–RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.

Identification of the gene encoding a type 1 RNase H with an N‐terminal double‐stranded RNA binding domain from a psychrotrophic bacterium

The gene encoding a bacterial type 1 RNase H, termed RBD‐RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD‐RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD‐RNase HI has a double‐stranded RNA binding domain (RBD) at the N‐terminus, which is commonly present at the N‐termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD‐RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD‐RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD‐RNase HI showed poor cleavage‐site specificity for oligomeric substrates. SIB1 RBD‐RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD‐RNase HI. This is the first report on the biochemical characterization of RBD‐RNase HI.

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half‐lives of SIB1 and E. coli RNases HII at 30 °C were ∼ 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by ∼ 0.2 m and ∼ 5 °C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 °C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low‐ and high‐activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high‐ and low‐activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low‐ and high‐activity type RNases H, but these types are not correlated with RNase H families.