Refined X-ray Structure of Dictyostelium discoideum Nucleoside Diphosphate Kinase at 1.8 Å resolution

Moréra, Solange; Lebras, G.; Lascu, Ioan; Ml, Lacombe; Véron, Michel; Janin, Joël

doi:10.1006/jmbi.1994.1689

Cited by 64 publications

(74 citation statements)

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“…Data were treated as described in the Experimental Procedures and give (k cat /K M ) Im ATP ) 8 M -1 s -1 for the reaction of H122A‚Im with ATP ( Figure 7; Table 4A). 3 This rate constant is 5000-fold less than the value of FIGURE 6: Superposition of the H122A structure (colored by atom type) with wild-type NDPK (in green) (16). Both structures were solved in the absence of nucleotide.…”

Section: Differential Effect Of Substituents On the Reactivity Of Amimentioning

confidence: 97%

Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis^,

et al. 2000

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We have explored the ability of a nucleoside diphosphate kinase (NDPK) mutant in which the nucleophilic histidine has been replaced by glycine (H122G) to transfer phosphate from ATP to alcohols of varying pK a , size, shape, and polarity. This cavity mutant does indeed act as a primitive alcohol kinase. The rate of its phosphoryl transfer to alcohols varies considerably, with values spanning a ∆∆G ‡ range of 4 kcal/mol, whereas the alcohols have very similar intrinsic reactivities. Analysis of these results suggests that the ability to carry out phosphoryl transfer within the cavity is not a simple function of being small enough to enter the cavity, but rather is a complex function of steric, solvation, entropic, van der Waals packing, and electrostatic properties of the alcohol. In addition, large differences are observed between the reactivities of alcohols within the nucleophile cavity of H122G and the reactivities of the same alcohols within the nucleophile cavity of H122A, a mutant NDPK that differs from H122G by a single methyl group within the cavity. The crystal structures of the two cavity mutants are very similar to one another and to wild-type NDPK, providing no evidence for a structurally perturbed active site. The differences in reactivity between the two mutant proteins illustrate a fundamental limitation of energetic analysis from site-directed mutagenesis: although removal of a side chain is generally considered to be a conservative change, the energetic effects of any given mutation are inextricably linked to the molecular properties of the created cavity and the surrounding protein environment.Previous studies of small molecule rescue of either the reactivity or stability of cavity mutants have provided insights into protein energetics. Mutagenesis of the lysine general base of aspartate aminotransferase to an alanine allowed Toney and Kirsch to investigate the ability of exogenous amines to restore catalytic activity to this cavity mutant (7). Matthews and co-workers demonstrated that a variety of small, mainly nonpolar ligands stabilize a cavity mutant of T4 lysozyme (8-10). In both studies, the authors obtained an energetic picture of the primarily hydrophobic cavities that were investigated by varying the small molecule that occupied those cavities. These earlier results suggested that a systematic study of rescue in a more complex cavity that is accessible to solvent and bordered by charged residues could provide information about the energetic features of protein crevices such as those often present at active sites.We have explored the energetic features of such a protein cavity using H122G, a site-directed mutant of nucleoside diphosphate kinase (NDPK) 1 in which the nucleophilic histidine has been replaced by a glycine. Wild-type NDPK interconverts NTPs and NDPs by catalyzing successive phosphoryl transfers, first transferring a phosphoryl group from an NTP to histidine 122 to form a phosphorylated enzyme and an NDP, then catalyzing a second transfer between the phosphorylated...

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Section: Differential Effect Of Substituents On the Reactivity Of Amimentioning

confidence: 97%

Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis^,

et al. 2000

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“…In the cubic crystal form, the asymmetric unit contains a dimer, and the hexamer is reconstituted by applying a 3-fold crystal symmetry. The subunits have the characteristic fold shared by all other NDP kinases and based on a four-stranded antiparallel ␤-sheet with five connecting ␣-helices (43). The most significant difference between Nm23-H4 and other NDP kinases is at the C terminus, in which the last 12 residues are disordered and cannot be located in the electron density map.…”

Section: Fig 2 Autophosphorylation Of Wild-type and Mutantmentioning

confidence: 99%

The Human nm23-H4 Gene Product Is a Mitochondrial Nucleoside Diphosphate Kinase

Milon¹,

Meyer²,

Chiadmi³

et al. 2000

Journal of Biological Chemistry

133

129

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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 fulllength protein and as a truncated form missing the Nterminal 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.

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“…8 The Kpn loop, located near the threefold axis of the hexamer, is heavily involved in subunit contacts. 9 The carboxylate of C-terminal Ala136 is firmly held in place by a salt bridge with Arg4, effectively closing the polypeptide chain on itself. In the human protein, the chain is 15 residues longer, and the C-terminal Glu152 interacts with another subunit in the hexamer.…”

mentioning

confidence: 99%

“…This is similar to the 640 Å 2 buried in Dictyostelium NDP kinase, but represents only 60% of the area buried in the human or Drosophila homologs. 9 The trimer contacts around the threefold axis are also less extensive than in the eukaryotic proteins, burying 1140 Å 2 per subunit instead of 1700 Å 2 in Drosophila. In either case, the eukaryotic C-terminal extension makes up for the difference.…”

mentioning

confidence: 99%

“…The dimer interfaces contain three hydrogen bonds, the trimer interfaces, five per subunit, fewer than in the eukaryotic proteins, where the C-terminal extension makes additional polar interactions. 9 On the other hand, a salt bridge is formed by Arg80 and Asp93 within trimers. This interaction is specific to M. tuberculosis, neutral side-chains occupying positions equivalent to 80 and 93 in most other NDP kinase sequences.…”

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confidence: 99%

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X‐ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase

Chen¹,

Moréra²,

Mocan³

et al. 2002

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Refined X-ray Structure of Dictyostelium discoideum Nucleoside Diphosphate Kinase at 1.8 Å resolution

Cited by 64 publications

References 0 publications

Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis^,

Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis^,

The Human nm23-H4 Gene Product Is a Mitochondrial Nucleoside Diphosphate Kinase

X‐ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase

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Refined X-ray Structure of Dictyostelium discoideum Nucleoside Diphosphate Kinase at 1.8 Å resolution

Cited by 64 publications

References 0 publications

Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis,

Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis,

The Human nm23-H4 Gene Product Is a Mitochondrial Nucleoside Diphosphate Kinase

X‐ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase

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Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis^,

Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant: A Cautionary Tale for Site-Directed Mutagenesis^,