Structural and Biophysical Analysis of the DNA Binding Properties of Myelin Transcription Factor 1

Abstract

Zinc binding domains, or zinc fingers (ZnFs), form one of the most numerous and most diverse superclasses of protein structural motifs in eukaryotes. Although our understanding of the functions of several classes of these domains is relatively well developed, we know much less about the molecular mechanisms of action of many others. Myelin transcription factor 1 (MyT1) type ZnFs are found in organisms as diverse as nematodes and mammals and are found in a range of sequence contexts. MyT1, one of the early transcription factors expressed in the developing central nervous system, contains seven MyT1 ZnFs that are very highly conserved both within the protein and between species. We have used a range of biophysical techniques, including NMR spectroscopy and data-driven macromolecular docking, to investigate the structural basis for the interaction between MyT1 ZnFs and DNA. Our data indicate that MyT1 ZnFs recognize the major groove of DNA in a way that appears to differ from other known zinc binding domains.

A Structural Analysis of DNA Binding by Myelin Transcription Factor 1 Double Zinc Fingers

“…3, A and B) reveals the sequence AAAGTTCA (palindromic sequence is underlined) as the F4F5 interaction surface. These data are in good agreement with our SPR and NMR analysis of the F5-DNA interaction (14), which showed that F5 has a footprint on the DNA that comprises the smaller AGT motif.…”

“…We recently assessed the binding of MyT1 to DNA and showed that the fifth zinc finger can fit into the DNA major groove and make contacts with the central AGT of the consensus sequence (14). Our data also indicated that double-finger constructs of both the two-ZF and the four-ZF clusters (F1F2, F4F5, F5F6, and F6F7) interact with the full AAAGTT consensus site with a higher affinity than does a single finger.…”

“…Previously, we experienced problems with degradation and intermediate chemical exchange during NMR studies of the double-finger proteins (14). We therefore optimized our experimental conditions (e.g.…”

“…A total of 10 different starting orientations between F4F5 and the DNA were chosen as starting structures for the docking. Sequences shown to be disordered in our previous NMR analysis, namely residues 799 -800 (N-terminal) and 872-873 (C-terminal), were defined as fully flexible during the calculations, as was the internal linker (828 -845) (14). Ambiguous interaction restraints for both the protein and the DNA were chosen based both on our NMR data from Figs.…”

“…Chemical shift changes were calculated as a weighted average of HN, N, and C␣ changes, using a previously reported equation (17,18). Assignments of the DNA alone were obtained from our previous study (14). One-bond HN residual dipolar couplings (RDCs) were recorded for the F4F5-DNA complex in NMR buffer containing 22.2 mg/liter Pf1 phage (ASLA Biotech), using the in-phase/ anti-phase pulse sequence (19).…”