Quantum Beam Science Research

5-9Developing a Technique for Operating a Protein Assembly=Identification of the Key Residue Controlling Protein Assembly via X-ray Crystallography=(a)The E134A mutant HaNDK tetramer
The wild-type HaNDK dimers superposed on
the E134A HaNDK tetramer
Electrostatic repulsion between oxygen atoms

Oxygen atom
E137

(b)
E134

Dimer / dimer interface

E134

E137

Oxygen atom

Fig.5-19 Three - dimensional structures HaNDKs(a) wild-type HaNDK dimer, and (b) E134A mutant
HaNDK tetramer. Orange circles indicate thelocation of the 134th residue responsible fortetrameric association.

In some proteins, the formation of a higher-orderedassembly is essential for enabling their biological function.
Such an assembly formation is known to be effective for
improving the stability and function of proteins. To develop atechnique for operating protein assembly, we attempted tochange the assembly state of nucleoside diphosphate kinase(NDK).
NDK exists in the cells of various organisms, and while
NDKs from most organisms assemble into a tetramer or ahexamer, NDK from the halophilic bacteria Halomonas
sp.593 (HaNDK) exceptionally forms a dimer. To identify thereason for the lack of dimer-dimer association of wild-type
HaNDK, its three-dimensional structure was determined via
X-ray crystallography (Fig.5-19(a)). It was found that, when
the structure of the wild-type HaNDK was superposed on thestructure of a known tetrameric NDK, the 134th and 137th
glutamic acids (E134, E137) directly contacted the E134 andE137 positions in the neighboring HaNDK molecule (Fig.5-20).
The fact that the overlay provides at least four negative
charges at the interface of the two dimer units suggests thatthe electrostatic repulsion prevents the dimer-dimer

Fig.5-20 The tetramer is the interface newly created by twodimer units of the E134A mutation
The wild-type HaNDK molecules (pink and blue sticks) aresuperposed on the E134A mutant HaNDK tetramer (black stick).
Bold sticks show the E134 and E137 glutamic acids of the wild-type
HaNDK.

association.
Given these results, we expect that the HaNDK dimer will
be associated into a higher-order assembly form if the E134
glutamic acid is replaced with alanine (E134A), which has anuncharged short side chain. In fact, the mutation of E134 toalanine (E134A mutant HaNDK) resulted in conversion ofthe assembly state of HaNDK from a dimer to a tetramer, asshown in (Fig.5-19(b)). Moreover, the specific activity of theHaNDK tetramer was 19% higher than that of the HaNDKdimer.
This study demonstrated that the assembly state of aprotein can be controlled with a minimum mutation if
detailed structural information is available. The investigationof the structural differences in the dimeric and tetramericassemblies will provide useful information for creating
higher-ordered assemblies, which will contribute to thecreation of proteins with increased enzymatic activity andstability.
Our research was accomplished with the support ofKagoshima University.

Reference
Arai, S. et al., A Structural Mechanism for Dimeric to Tetrameric Oligomer Conversion in Halomonas sp. Nucleoside Diphosphate Kinase, Protein Science,
vol.21, issue 4, 2012, p.498-510.

74 JAEA R&D Review 2012