Diversity in Structure and Function of the Ets Family PNT Domains Cameron D. Mackereth†, Manuela Scha ¨rpf†, Lisa N. Gentile Scott E. MacIntosh, Carolyn M. Slupsky and Lawrence P. McIntosh* Department of Biochemistry and Molecular Biology Department of Chemistry and The Biotechnology Laboratory, University of British Columbia, Vancouver BC, Canada V6T 1Z3 The PNT (or Pointed) domain, present within a subset of the Ets family of transcription factors, is structurally related to the larger group of SAM domains through a common tertiary arrangement of four a-helices. Previous studies have shown that, in contrast to the PNT domain from Tel, this domain from Ets-1 contains an additional N-terminal helix integral to its folded structure. To further investigate the structural plasticity of the PNT domain, we have used NMR spectroscopy to characterize this domain from two additional Ets proteins, Erg and GABPa. These studies both define the conserved and variable features of the PNT domain, and demonstrate that the additional N-terminal helix is also present in GABPa, but not Erg. In contrast to Tel and Yan, which self-associate to form insoluble polymers, we also show that the isolated PNT domains from Ets-1, Ets-2, Erg, Fli-1, GABPa, and Pnt-P2 are monomeric in solution. Furthermore, these soluble PNT domains do not associate in any pair-wise combination. Thus these latter Ets family PNT domains likely mediate interactions with additional components of the cellular signaling or transcriptional machinery. q 2004 Elsevier Ltd. All rights reserved. Keywords: transcription factor; SAM domain; protein interactions; Erg; GABP *Corresponding author Introduction Proteins involved in gene regulation and signal transduction are typically composed of a series of modular domains that confer a variety of binding and catalytic functions. As an example, components of signaling cascades use modules such as the SH2, SH3, and PDZ domains to regulate protein–protein association through the binding of specific peptide sequences. 1,2 Transcription factors contain a similar combinatorial assembly of domains, with functions including DNA recognition, transactivation, and auto-regulation. For example, the DNA-binding winged helix-turn-helix ETS domain defines the members of the Ets family of transcription factors. 3–5 A conserved PNT (or Pointed) domain is also found within a subset of the Ets transcription factors, including mammalian Ets-1, Ets-2, Erg, Fli-1, GABPa, and Tel, as well as Drosophila Pnt-P2 and Yan. First identified during cloning of the Pnt- P2 gene product in Drosophila 6 and characterized structurally through the NMR analysis of Ets-1, 7 the PNT domain has since been encompassed by the closely related family of SAM domains. These latter domains are found in numerous proteins involved in eukaryotic developmental and signal transduc- tion pathways (Figure 1). 8–10 Following the suggested nomenclature of the SMART 11 and SCOP 12 databases, the PNT domain distinction is restricted to members of the Ets family of transcrip- tion factors and represents a specific category within the larger set of SAM domains. 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. † C.D.M. & M.S. contributed equally to this work Present address: L. N. Gentile, Department of Chemistry, WesternWashington University, Bellingham, WA 98225, USA. Abbreviations used: CD, circular dichroism; ESI-MS, electrospray ionization mass spectrometry; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum correlation; MWCO, molecular weight cut-off; rms, root- mean-square; T m , midpoint thermal unfolding temperature; protein fragments are denoted in superscripted parentheses by the initial and final residue number from the native sequence, and if present, an asterisk (*) to indicate an N-terminal GSH or GSHM sequence remaining after thrombin cleavage of a His 6 affinity tag. E-mail address of the corresponding author: mcintosh@otter.biochem.ubc.ca doi:10.1016/j.jmb.2004.07.094 J. Mol. Biol. (2004) 342, 1249–1264