Altering the Double-Stranded DNA Specificity of the bZIP Domain of Zta with Site-Directed Mutagenesis at N182

Sreejana Ray; Desiree Tillo; Nima Assad; Aniekanabasi Ufot; Aleksey Porollo; Stewart R Durell; Charles Vinson

doi:10.1021/acsomega.1c04148

Altering the Double-Stranded DNA Specificity of the bZIP Domain of Zta with Site-Directed Mutagenesis at N182

ACS Omega. 2021 Dec 28;7(1):129-139. doi: 10.1021/acsomega.1c04148. eCollection 2022 Jan 11.

Authors

Sreejana Ray¹, Desiree Tillo^{1

2}, Nima Assad¹, Aniekanabasi Ufot¹, Aleksey Porollo^{3

4}, Stewart R Durell⁵, Charles Vinson¹

Affiliations

¹ Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 5000, Building 37, Bethesda, Maryland 20892, United States.
² Cancer Genetics Branch, National Cancer Institute, National Institutes of Health, Building 37, Bethesda, Maryland 20892, United States.
³ Center for Autoimmune Genomics and Etiology, Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, United States.
⁴ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, United States.
⁵ Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Building 37, Bethesda, Maryland 20892, United States.

Abstract

Zta, the Epstein-Barr virus bZIP transcription factor (TF), binds both unmethylated and methylated double-stranded DNA (dsDNA) in a sequence-specific manner. We studied the contribution of a conserved asparagine (N182) to sequence-specific dsDNA binding to four types of dsDNA: (i) dsDNA with cytosine in both strands ((DNA(C|C)), (ii, iii) dsDNA with 5-methylcytosine (5mC, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand ((DNA(5mC|C) and DNA(5hmC|C)), and (iv) dsDNA with methylated cytosine in both strands in all CG dinucleotides ((DNA(5mCG)). We replaced asparagine with five similarly sized amino acids (glutamine (Q), serine (S), threonine (T), isoleucine (I), or valine (V)) and used protein binding microarrays to evaluate sequence-specific dsDNA binding. Zta preferentially binds the pseudo-palindrome TRE (AP1) motif (T^-4G^-3A^-2G/_C ⁰T²C³A⁴ ). Zta (N182Q) changes binding to A³ in only one half-site. Zta(N182S) changes binding to G³ in one or both halves of the motif. Zta(N182S) and Zta(N182Q) have 34- and 17-fold weaker median dsDNA binding, respectively. Zta(N182V) and Zta(N182I) have increased binding to dsDNA(5mC|C). Molecular dynamics simulations rationalize some of these results, identifying hydrogen bonds between glutamine and A³ , but do not reveal why serine preferentially binds G³ , suggesting that entropic interactions may mediate this new binding specificity.