Point Mutations in awd Which Revert the Prune/Killer of Prune Lethal Interaction Affect Conserved Residues That Are Involved in Nucleoside Diphosphate Kinase Substrate Binding and Catalysis

Abstract

The awd gene of Drosophila melanogaster encodes a nucleoside diphosphate kinase. Killer of prune (Kpn) is a mutation in the awd gene which substitutes Ser for Pro at position 97 and causes dominant lethality in individuals that do not have a functional prune gene. This lethality is not due to an inadequate amount of nucleoside diphosphate (NDP) kinase activity. In order to understand why the prune/Killer of prune combination is lethal, even in the presence of an adequate NDP kinase specific activity level, and to understand the biochemical basis for the conditional lethality of the awd Kpn mutation, we generated second site mutations which revert this lethal interaction. All of the 12 revertants we recovered are second site mutations of the awd Kpn gene. Three revertants have deletions of the awd Kpn protein coding region. Two revertants have substitutions of the initiator methionine and do not accumulate KPN protein. Seven revertants have amino acid substitutions of conserved residues that are likely to affect the active site: five of these have no enzymatic activity and two have a very low level of specific activity. These data suggest that an altered NDP kinase activity is involved in the mechanism underlying the conditional lethality of the awd Kpn mutation.

Loss-of-Function Mutations in a Glutathione S-Transferase Suppress the prune-Killer of prune Lethal Interaction

“…We discovered that this vital gene is awd (Biggs et al 1988). Kpn is actually a missense mutation that causes a substitution of Ser for Pro at position 97 of the AWD protein (Lascu et al 1992;Timmons et al 1995). We renamed Kpn as awd Kpn .…”

“…The awd gene encodes a single 0.8-kb transcript (Dearolf et al 1988b) that is translated into a 17-kDa subunit of a 100-kDa homo-hexameric protein that accounts for 98% of the nucleoside diphosphate kinase activity of larval extracts (Biggs et al 1990;Timmons et al 1995). Nucleoside diphosphate kinases can transfer the terminal phosphate from any nucleoside triphosphate donor to any nucleoside diphosphate acceptor via a ping-pong mechanism.…”

“…A 15% SDS-PAGE gel was loaded with the equivalent of three larvae per well in 23 SDS sample buffer. Purified AWD polyclonal antibody previously generated in the laboratory (Timmons et al 1995) was used at 1:750 to detect the 15-kDa monomers of AWD Kpn . The blot was normalized to b-tubulin (E7: Developmental Studies Hybridoma Bank) at 1:500.…”

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“…Histidine 118 is the site of NME autophosphorylation, and the H118F transfectants in the originally published MDA-MB-435 cell line failed to show NDPK or HPK activity above vector controls [41]. P96 is located on a loop structure of NME and is known as the killer of prune mutation due to its lethal interaction with the Drosophila prune gene [42][43][44]. Based on crystal structure and developmental studies in Drosophila, it is speculated that P96S mutation causes a change in NME conformation which possibly affects its binding partners or oligomerization leading to compromised/partial function.…”

Nucleoside diphosphate kinases (NDPKs) in animal development

“…In the 1990s, it was eventually realized that K-pn in fact is an allele of awd (and was hence renamed awd K-pn ), which carries a proline to serine substitution in position 97 (which corresponds to P96 in NM23-H1/2) that ensures structural integrity of the multimeric forms of NDPK [36,37]. It is thought that the K-pn (P97S) mutation lies in a mutant loop that imparts an increased flexibility but reduced stability to NDPK protein's hexameric quaternary structure.…”