Bagautdin Bagautdinov

Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 °C

We found that the CutA1 protein, from Pyrococcus horikoshii (PhCutA1), has an extremely high denaturation temperature (T d) of nearly 150 °C, which exceeds the highest record determined by DSC by about 30 °C. To elucidate the mechanism of the ultra‐high stability of PhCutA1, we analyzed the crystal structures of CutA1 proteins from three different sources, P. horikoshii, Thermus thermophilus, and Escherichia coli, with different growth temperatures (98, 75, and 37 °C). This analysis revealed that the remarkably increased number of ion pairs in the monomeric structure contributes to the stabilization of the trimeric structure and plays an important role in enhancing the T d, up to 150 °C, for PhCutA1.

Protein biotinylation visualized by a complex structure of biotin protein ligase with a substrate

Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL·BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5′-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5′-AMP, thereby providing the complex crystals. The four crystallographically independent BPL·BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction.

Structure of indole-3-glycerol phosphate synthase from Thermus thermophilus HB8: implications for thermal stability

The three-dimensional structure of indole-3-glycerol phosphate synthase (IGPS) from the thermophilic bacterium Thermus thermophilus HB8 (TtIGPS) has been determined at 1.8 Å resolution. The structure adopts a typical (β/α)(8)-barrel fold with an additional N-terminal extension of 46 residues. A detailed comparison of the crystal structure of TtIGPS with available structures of IGPS from the archaeon Sulfolobus solfataricus (SsIGPS) and the bacteria Thermotoga maritima (TmIGPS) and Escherichia coli (EcIGPS) has been performed. Although the overall folds of the proteins are the same, there are differences in amino-acid composition, structural rigidity, ionic features and stability clusters which may account for the high thermostability of the hyperthermophilic (SsIGPS and TmIGPS) and thermophilic (TtIGPS) proteins when compared with the mesophilic EcIGPS. The thermostability of IGPS seems to be established mainly by favourable interactions of charged residues, salt bridges and the spatial distribution of relatively rigid clusters of extensively interacting residues.

Remarkable improvement in the heat stability of CutA1 from Escherichia coli by rational protein design

To enhance the heat stability of the CutA1 protein from Escherichia coli (EcCutA1) so that it has comparable stability to CutA1 from Pyrococcus horikoshii with a denaturation temperature (T(d)) of 150°C, we used the Stability Profile of Mutant Protein (SPMP) to examine the structure-sequence (3D-1D) compatibility between the conformation of EcCutA1 and its native sequence [J. Mol. Biol., 248, 733-738, (1995)]. We identified seven residues in EcCutA1 that were incompatible in terms of dihedral angles and hydrophobicity. These residues were replaced with appropriate amino acids, and the mutant proteins were evaluated for changes in stability by DSC and denaturant denaturation. The mutations that were introduced at five out of the seven positions improved the stability of EcCutA1. The T(d) values of single (S11A) and triple (S11V/E61V/Q73V) mutants improved by 16.5 and 26.6°C, respectively, compared to that of the wild-type protein (89.9°C). These analyses showed that (1) the stability of EcCutA1 is remarkably improved by slight substitutions, even though the stability of the wild-type protein is considerably high, (2) remarkable improvements in the stability can be quantitatively explained based on the newly solved native structure, and (3) SPMP is a powerful tool to examine substitutions that improve protein stability.

Thermodynamic analysis of unusually thermostable CutA1 protein from human brain and its protease susceptibility

Unusually stable proteins are a disadvantage for the metabolic turnover of proteins in cells. The CutA1 proteins from Pyrococcus horikoshii and from Oryza sativa (OsCutA1) have unusually high denaturation temperatures (Td) of nearly 150 and 100 °C, respectively, at pH 7.0. It seemed that the CutA1 protein from the human brain (HsCutA1) also has a remarkably high stability. Therefore, the thermodynamic stabilities of HsCutA1 and its protease susceptibility were examined. The Td was remarkably high, being over 95 °C at pH 7.0. The unfolding Gibbs energy (ΔG(0)H2O) was 174 kJ/mol at 37 °C from the denaturant denaturation. The thermodynamic analysis showed that the unfolding enthalpy and entropy values of HsCutA1 were considerably lower than those of OsCutA1 with a similar stability to HsCutA1, which should be related to flexibility of the unstructured properties in both N- and C-terminals of HsCutA1. HsCutA1 was almost completely digested after 1-day incubation at 37 °C by subtilisin, although OsCutA1 was hardly digested at the same conditions. These results indicate that easily available fragmentation of HsCutA1 with remarkably high thermodynamic stability at the body temperature should be important for its protein catabolism in the human cells.

Crystallization and preliminary X‐ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein-protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPDeltaN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 complexes as well as crystals of BPL*, BPL** and BCCPDeltaN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 crystals were collected at 100 K to 2.7 and 2.0 A resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2(1), with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 A, beta = 95.9 degrees . Assuming two subunits of the complex per asymmetric unit gives a V(M) value of 2.45 A(3) Da(-1) and a solvent content of 50%.

The structures of the CutA1 proteins from Thermus thermophilus and Pyrococcus horikoshii: characterization of metal-binding sites and metal-induced assembly

CutA1 (copper tolerance A1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. In Escherichia coli it is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X-ray structures of CutA1 from the thermophilic bacterium Thermus thermophilus (TtCutA1) with and without bound Na(+) at 1.7 and 1.9 Å resolution, respectively, and from the hyperthermophilic archaeon Pyrococcus horikoshii (PhCutA1) in complex with Na(+) at 1.8 Å resolution have been determined. Both are short and rigid proteins of about 12 kDa that form intertwined compact trimers in the crystal and solution. The main difference in the structures is a wide-type β-bulge on top of the TtCutA1 trimer. It affords a mechanism for lodging a single-residue insertion in the middle of β2 while preserving the interprotomer main-chain hydrogen-bonding network. The liganded forms of the proteins provide new structural information about the metal-binding sites and CutA1 assembly. The Na(+)-TtCutA1 structure unveils a dodecameric assembly with metal ions in the trimer-trimer interfaces and the lateral clefts of the trimer. For Na(+)-PhCutA1, the metal ion associated with six waters in an octahedral geometry. The structures suggest that CutA1 may contribute to regulating intracellular metal homeostasis through various binding modes.

Structure of putative CutA1 from Homo sapiens determined at 2.05 Å resolution

The structure of human brain CutA1 (HsCutA1) has been determined using diffraction data to 2.05 A resolution. HsCutA1 has been implicated in the anchoring of acetylcholinesterase in neuronal cell membranes, while its bacterial homologue Escherichia coli CutA1 is involved in copper tolerance. Additionally, the structure of HsCutA1 bears similarity to that of the signal transduction protein PII, which is involved in regulation of nitrogen metabolism. Although several crystal structures of CutA1 from various sources with different rotation angles and degrees of interaction between trimer interfaces have been reported, the specific functional role of CutA1 is still unclear. In this study, the X-ray structure of HsCutA1 was determined in space group P2(1)2(1)2(1), with unit-cell parameters a = 68.69, b = 88.84, c = 125.33 A and six molecules per asymmetric unit. HsCutA1 is a trimeric molecule with intertwined antiparallel beta-strands; each subunit has a molecular weight of 14.6 kDa and contains 135 amino-acid residues. In order to obtain clues to the possible function of HsCutA1, its crystal structure was compared with those of other CutA1 and PII proteins.

Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3

Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 angstroms resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3(2)21, with unit-cell parameters a = b = 96.06, c = 298.90 angstroms. Assuming the presence of one hexamer in the asymmetric unit gives a V(M) value of 2.36 angstroms3 Da(-1) and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 angstroms resolution.