Dynamic conformations of nucleophosmin (NPM1) at a key monomer-monomer interface affect oligomer stability and interactions with granzyme B

Wei D. Duan-Porter, Virgil L. Woods, Kimberly D. Maurer, Sheng Li, Antony Rosen

Research output: Contribution to journalArticle

Abstract

Nucleophosmin (NPM1) is an abundant, nucleolar tumor antigen with important roles in cell proliferation and putative contributions to oncogenesis. Wild-type NPM1 forms pentameric oligomers through interactions at the amino-terminal core domain. A truncated form of NPM1 found in some hepatocellular carcinoma tissue formed an unusually stable oligomer and showed increased susceptibility to cleavage by granzyme B. Initiation of translation at the seventh methionine generated a protein (M7-NPM) that shared all these properties. We used deuterium exchange mass spectrometry (DXMS) to perform a detailed structural analysis of wild-type NPM1 and M7-NPM, and found dynamic conformational shifts or local ''unfolding'' at a specific monomer-monomer interface which included the β-hairpin ''latch.'' We tested the importance of interactions at the β-hairpin ''latch'' by replacing a conserved tyrosine in the middle of the β-hairpin loop with glutamic acid, generating Y67E-NPM. Y67E-NPM did not form stable oligomers and further, prevented wild-type NPM1 oligomerization in a dominant-negative fashion, supporting the critical role of the b-hairpin ''latch'' in monomer-monomer interactions. Also, we show preferential cleavage by granzyme B at one of two available aspartates (either D161 or D122) in M7-NPM and Y67E-NPM, whereas wild-type NPM1 was cleaved at both sites. Thus, we observed a correlation between the propensity to form oligomers and granzyme B cleavage site selection in nucleophosmin proteins, suggesting that a small change at an important monomer-monomer interface can affect conformational shifts and impact proteinprotein interactions.

Original languageEnglish (US)
Article numbere115062
JournalPloS one
Volume9
Issue number12
DOIs
StatePublished - Dec 9 2014

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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