Zinc finger motif for single‐stranded nucleic acids? investigations by nuclear magnetic resonance

Abstract

Nuclear magnetic resonance (NMR) methods have been used to address issues regarding the relevance and feasibility of zinc binding to “zinc finger‐like” sequences of the type C‐X₂‐C‐X₄‐H‐X₄‐C [referred to as CCHC or retroviral‐type (RT) zinc finger sequences]. One‐dimensional (1D) NMR experiments with an 18‐residue synthetic peptide containing the amino acid sequence of an HIV‐1 RT‐zinc finger domain (HIV1‐F1) indicate that the sequences are capable of binding zinc tightly and stoichiometrically. ¹H‐¹¹³Cd spin echo difference NMR data confirm that the Cys and His amino acids are coordinated to metal in the ¹¹³Cd adduct. The 3D structure of the zinc adduct [Zn(HIV1‐F1)] was determined to high atomic resolution by a new NMR‐based approach that utilizes 2D‐NOESY back‐calculations as a measure of the consistency between the structures and the experimental data. Several interesting structural features were observed, including (1) the presence of extensive internal hydrogen bonding, and (2) the similarity of the folding of the first six residues to the folding observed by X‐ray crystallography for related residues in the iron domain of rubredoxin. Structural constraints associated with conservatively substituted glycines provide further rationale for the physiological relevance of the zinc adduct. Similar NMR and structural results have been obtained for the second HIV‐1 RT‐zinc finger peptide, Zn(HIV1‐F2). NMR studies of the zinc adduct with the NCP isolated directly from HIV‐1 particles provide solid evidence that zinc finger domains are formed that are conformationally similar (if not identical) to the peptide structures. The motif has been found in several other single‐stranded nucleic acid binding proteins, including a human protein, and may represent a common motif analogous to the “classical” zinc finger motif widely distributed in duplex‐DNA binding proteins.