Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability. ß

The fully human monoclonal antibody KMTR2 acts as a strong direct agonist for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), which is capable of inducing apoptotic cell death without cross-linking. To investigate the mechanism of direct agonistic activity induced by KMTR2, the crystal structure of the extracellular region of TRAIL-R2 and a Fab fragment derived from KMTR2 (KMTR2-Fab) was determined to 2.1 Å resolution. Two KMTR2-Fabs assembled with the complementarity-determining region 2 of the light chain via two-fold crystallographic symmetry, suggesting that the KMTR2-Fab assembly tended to enhance TRAIL-R2 oligomerization. A single mutation at Asn53 to Arg located at the two-fold interface in the KMTR2 resulted in a loss of its apoptotic activity, although it retained its antigen-binding activity. These results indicate that the strong agonistic activity, such as apoptotic signaling and tumor regression, induced by KMTR2 is attributed to TRAIL-R2 superoligomerization induced by the interdimerization of KMTR2.

Light scattering and chemical cross-linking analyses of nucleoside diphosphate kinase (NDK) from moderate halophile, Halomonas sp. 593 (HaNDK), unambiguously demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK (PaNDK), a nonhalophilic counterpart, and other NDKs from Gram-negative bacteria, which all formed a tetrameric structure. Comparison of HaNDK and PaNDK showed that the HaNDK was less thermally stable than the PaNDK: the optimum temperature of PaNDK enzyme activity was 20 degrees C higher than that of HaNDK. However, the HaNDK readily refolded and reassembled back to the active dimeric structure, upon heat denaturation at 0.2 M NaCl, as soon as the temperature was lowered. On the contrary, the thermally more stable PaNDK was irreversibly denatured at its melting temperature.

Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium. However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition. Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation. The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations. This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation.

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1-65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 Å resolution with space groups of P2(1) and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.