Role of surface residues on defining the functional specificity of Nm23 metastasis suppressors

Members of the human Nm23 family (also called NME) act as metastasis suppressors and they exhibit multiple enzymatic activities including phosphorylation of nucleoside diphosphates and DNA cleavage. Although highly similar, the two most extensively studied members, Nm23-H1 and Nm23-H2, display differential preferences for protein interactions (1). The resolved structures of Nm23-H1/2 revealed that several clusters of isoform-specific amino acids are located on exposed surfaces of these proteins.

Aims. To assess the role of surface clusters on Nm23-H1/2 as determinants of protein recognition.

Methods. Specific surface amino acids were swapped between Nm23-H1 and Nm23-H2 by site-directed mutagenesis. Wild-type and mutants were examined by co-immunoprecipitation (Co-IP) assays and functional characterizations in transfected cell lines.

Results. Nm23-H1 mutants with multiple mutations were generated: Q42R/H69N, Q42R/A62P/H69N, E124K/G131S, E124K/G131S/H135K, Q147H/N148D, T143K/Q147H/N148D, and L47H/E50Q. The mutants were successfully expressed in HEK293 and MDA-MB-231 cell lines. Protein partners of Nm23 showed differential binding preferences for wild-type Nm23-H1/2 and Nm23-H1 mutants.

Discussion. The successful expression of Nm23-H1 mutants in transfected cells suggests that the alteration of these outer surface residues did not significantly affect the overall structure of Nm23-H1. Preliminary data suggested that both Nm23-H1 and H2 could form protein complexes with G in Co-IP assay, but show different affinities for the A kinase anchor protein Lbc. Whether the surface clusters can play any role in determining the functions of Nm23 isoforms remains to be uncovered.