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Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors.

ABSTRACT: The Ndt80 protein of the yeast Saccharomyces cerevisiae is the founding member of a new sub-family of proteins in the Ig-fold superfamily of transcription factors. The crystal structure of Ndt80 bound to DNA shows that it makes contacts through several loops on one side of the protein that connect beta-strands which form the beta-sandwich fold common to proteins in this superfamily. However, the DNA-binding domain of Ndt80 is considerably larger than many other members of the Ig-fold superfamily and it appears to make a larger number of contacts with the DNA than these proteins. To determine the contribution of each of these contacts and to examine if the mechanism of Ndt80 DNA binding was similar to other members of the Ig-fold superfamily, amino acid substitutions were introduced at each residue that contacts the DNA and assayed for their effect on Ndt80 activity. Many of the mutations caused significant decreases in DNA-binding affinity and transcriptional activation. Several of these are in residues that are not found in other sub-families of Ig-fold proteins. These additional contacts are likely responsible for Ndt80's ability to bind DNA as a monomer while most other members require additional domains or cofactors to recognize their sites.

SUBMITTER: Fingerman IM

PROVIDER: S-EPMC419620 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors.

Fingerman Ian M IM Sutphen Kristina K Montano Sherwin P SP Georgiadis Millie M MM Vershon Andrew K AK

Nucleic acids research 20040525 9

The Ndt80 protein of the yeast Saccharomyces cerevisiae is the founding member of a new sub-family of proteins in the Ig-fold superfamily of transcription factors. The crystal structure of Ndt80 bound to DNA shows that it makes contacts through several loops on one side of the protein that connect beta-strands which form the beta-sandwich fold common to proteins in this superfamily. However, the DNA-binding domain of Ndt80 is considerably larger than many other members of the Ig-fold superfamily ...[more]

PMID: 15161958

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Project description:The Nipah virus (NiV) phosphoprotein (P) gene encodes four proteins. Three of these-P, V, and W-possess a common N-terminal domain but distinct C termini. These proteins interact with immune modulators. Previous studies demonstrated that P, V, and W bind STAT1 and STAT4 and that V also interacts with STAT2 but not with STAT3. The STAT1 and STAT2 interactions block interferon (IFN)-induced STAT tyrosine phosphorylation. To more fully characterize the interactions of P, V, and W with the STATs, we screened for interaction of each viral protein with STATs 1 to 6 by coimmunoprecipitation. We demonstrate that NiV P, V, and W interact with STAT4 through their common N-terminal domain and block STAT4 activity, based on a STAT4 response element reporter assay. Although none of the NiV proteins interact with STAT3 or STAT6, NiV V, but not P or W, interacts with STAT5 through its unique C terminus. Furthermore, the interaction of NiV V with STAT5 was not disrupted by overexpression of the N-terminal binding STAT1 or the C-terminal binding MDA5. NiV V also inhibits a STAT5 response element reporter assay. Residues 114 to 140 of the common N-terminal domain of the NiV P gene products were found to be sufficient to bind STAT1 and STAT4. Analysis of STAT1-STAT3 chimeras suggests that the P gene products target the STAT1 SH2 domain. When fused to GST, the 114-140 peptide is sufficient to decrease STAT1 phosphorylation in IFN-β-stimulated cells, suggesting that this peptide could potentially be fused to heterologous proteins to confer inhibition of STAT1- and STAT4-dependent responses.IMPORTANCE How Nipah virus (NiV) antagonizes innate immune responses is incompletely understood. The P gene of NiV encodes the P, V, and W proteins. These proteins have a common N-terminal sequence that is sufficient to bind to STAT1 and STAT2 and block IFN-induced signal transduction. This study sought to more fully understand how P, V, and W engage with the STAT family of transcription factors to influence their functions. The results identify a novel interaction of V with STAT5 and demonstrate V inhibition of STAT5 function. We also demonstrate that the common N-terminal residues 114 to 140 of P, V, and W are critical for inhibition of STAT1 and STAT4 function, map the interaction to the SH2 region of STAT1, and show that a fusion construct with this peptide significantly inhibits cytokine-induced STAT1 phosphorylation. These data clarify how these important virulence factors modulate innate antiviral defenses.

Dataset Information

Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors.

Publications

Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors.

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