Backbone dynamics of a short PU.1 ETS domain

Abstract

The resonance assignments, secondary structure and backbone dynamics of the ETS domain of the transcription factor PU.1 have been determined for the free protein in solution by NMR spectroscopy. The secondary structure for the free ETS domain is similar to that observed in the crystal structure of the PU.1 protein complexed with DNA, except that helix α2 and recognition helix α3 are shorter for the free protein in solution. Backbone dynamics of the protein have been examined using amide hydrogen-deuterium exchange and 15N laboratory-frame spin relaxation measurements. A significant probability of local unfolding of helix α2, which precedes the loop-helix-loop DNA recognition domain, is inferred from the very fast hydrogen-deuterium exchange for amide protons in this helix. The 15N relaxation measurements indicate that the protein is partially oligomerized at a concentration of 2.5 mM, but monomeric at a concentration of 0.3 mM. The 15N relaxation data for the low concentration sample were interpreted, using the model-free formalism, to provide insight into protein dynamics on picosecond-nanosecond and microsecond-millisecond time scales. High flexibility of the protein backbone is observed for the residues in the loop between α2 and α3. This loop is variable in length and in structure within the class of winged helix proteins and is partially responsible for binding to DNA. The dynamic properties observed for α2, α3 and the intervening loop may indicate a correlation between protein plasticity in particular structural elements and recognition of specific DNA sequences.