PDBsum entry: 1ehw

Transferase

PDB id: 1ehw

Contents

Protein chains

143 a.a. *

Ligands

SO4 ×3

Waters ×83

* Residue conservation analysis

PDB id:

1ehw

Name:

Transferase

Title:

Human nucleoside diphosphate kinase 4

Structure:

Nucleoside diphosphate kinase. Chain: a, b. Synonym: ndpk h4. Engineered: yes. Mutation: yes. Other_details: n-terminus truncated, n-terminal his tag

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Hexamer (from PDB file)

Resolution:

2.40Å R-factor: 0.184 R-free: 0.229

Authors:

L.Milon,P.Meyer,M.Chiadmi,A.Munier,M.Johansson,A.Karlsson,I. J.Capeau,J.Janin,M-L.Lacombe

Key ref:

L.Milon et al. (2000). The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase. J Biol Chem, 275, 14264-14272. PubMed id: 10799505 DOI: 10.1074/jbc.275.19.14264

Date:

23-Feb-00 Release date: 17-May-00

Protein chains

O00746  (NDKM_HUMAN) -  Nucleoside diphosphate kinase, mitochondrial

Seq: 187 a.a.

Struc: 143 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.4.6 - Nucleoside-diphosphate kinase.
• Reaction: ATP + nucleoside diphosphate = ADP + nucleoside triphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: nucleoside diphosphate phosphorylation (4 terms)
• Biochemical function: nucleoside diphosphate kinase activity (2 terms)

reference

DOI no: 10.1074/jbc.275.19.14264

J Biol Chem 275:14264-14272 (2000)

PubMed id: 10799505

The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase.

L.Milon, P.Meyer, M.Chiadmi, A.Munier, M.Johansson, A.Karlsson, I.Lascu, J.Capeau, J.Janin, M.L.Lacombe.

ABSTRACT

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.

Selected figure(s)

Figure 4.
Fig. 4. Nm23-H4 structure and comparison with NDP kinase B. A, the Nm23-H4 hexamer is viewed along the 3-fold axis. The front trimer is in blue, and the back trimer is in yellow. The boxed region is shown in detail in C. B, superposition of the H4 subunit in yellow and the NDP kinase B subunit with bound GDP in green. Differences between the two structures are localized at the N and C termini and on the sides of the nucleotide binding cleft formed by helices A- 2 and the K-pn loop. The latter is also involved in the subunit interface forming the trimers. C, details of subunit interactions in the back trimer (boxed region in A). Red balls are the carbonyl oxygens of Ser129, Gly139, and Ser142 of the K-pn loop interacting with the side chain of Arg64' from the neighboring subunit. The Ser129 side chain H-bonds to the carbonyl of His144 in the same subunit. In NDPK B (green bonds), the residue corresponding to Ser129 is Pro96. The C terminus, disordered in H4, makes subunit contacts in NDPK B, including a H-bond to Gln111 equivalent to His144 (drawn with Molscript (61) and Raster3D (62)). D, close-up of the proline-to-serine substitution at position 129. NDPK H4 is colored according to atom type and NDPK B is in green. In H4, two hydrogen bonds from the serine NH and OH cause a rotation of the carbonyl group of His144. The proline in NDPK B cannot make these bonds (drawn with TURBO).

Figure 7.
Fig. 7. Submitochondrial localization and membrane-bound association of Nm23-H4. A, submitochondrial localization of Nm23-H4: aliquots of each subfractions were analyzed by SDS-PAGE and revealed with the indicated antibodies. The aliquots were normalized to deposit an equivalent fraction of the initial purified mitochondria (20 µg). OM, outer membrane; IMS, intermembrane space; IM, inner membrane; Mx, matrix. B, membrane-bound association of Nm23-H4, after treatment with (+) or without ( ) alkali and centrifugation of the inner membrane of HEK 293 mitochondria. The pellet ( P) and the supernatant (S) were loaded and revealed by the anti-Nm23-H4 antibodies (Nm23-H4) or the anti-cytochrome oxidase subunit II (cyt. oxidase).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 14264-14272) copyright 2000.

Figures were selected by an automated process.