A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase

Abstract

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.

Crystal structure of Lamellipodin implicates diverse functions in actin polymerization and Ras signaling

“…Dimerization or higher-order oligomerization of protein molecules often plays an important role in enhancing their biological functions through an increased binding avidity (Stein et al, 2003;Shiroishi et al, 2006;Depetris et al, 2009;Arai et al, 2012;McElroy et al, 2012). To validate the biological relevancy of the two contact sites observed from the crystal structures, mutagenesis and gel filtration chromatography RESEARCH ARTICLE Protein Cell & the β5-β6 loop, likely due to the absence of the crucial Arg416 residue in the β1-β2 loop (Leu252 in Grb10).…”

Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

“…Whatever the quaternary architecture, NDPKs share a common-dimer unit. Recently, such dimer unit has been found in solution for the NDPK from moderately halophilic bacteria [17], [18]. As the subunit assembly is very different in hexameric and tetrameric NDPKs, the role of the quaternary structure for protein varies between the two types of NDPKs [19].…”

“…The importance of specific steric and electrostatic interactions in the dimer-dimer assembly of the NDPK from moderately halophilic bacteria has been established [18], [22]. One interaction present in Mt -NDPK but missing in other NDPKs, is the intersubunit salt bridge Arg 80 -Asp 93 located on the protein surface.…”

Effects of Mutations of Thermolysin, Asn116 to Asp and Asp150 to Glu, on Salt-Induced Activation and Stabilization

“…33) Human angiotensin-converting enzyme changes the position of an active-site loop in the presence of NaCl, and the binding of three Arg residues (Arg186, Arg489, and Arg522) with one chloride ion is responsible for this activation. 34) It appears that the mechanisms of salt-induced activation and stabilization of thermolysin are different from those of the halophilic enzymes mentioned above.…”