High-resolution structures of variant Zif268–DNA complexes: implications for understanding zinc finger–DNA recognition

Abstract

The protein-DNA contacts observed in these complexes reveal the basis for the specificity demonstrated by these Zif268 variants. Many, but not all, of the contacts can be rationalized in terms of a recognition code, but the predictive value of such a code is limited. The structures illustrate how modest changes in the docking arrangement accommodate the new sidechain-base and sidechain-phosphate interactions. Such adaptations help explain the versatility of naturally occurring zinc finger proteins and their utility in design.

Development of Zinc Finger Domains for Recognition of the 5′-ANN-3′ Family of DNA Sequences and Their Use in the Construction of Artificial Transcription Factors

“…Ideally, zinc finger proteins could be readily constructed to bind any DNA sequence; however, information regarding zinc finger/DNA interactions is constrained to just a few of the 64 possible 5Ј-NNN-3Ј DNA subsites. Structural analysis of several domains that may specify a 5Ј-nucleotide other than guanine has not revealed any specific interaction from position 6 of the ␣-helix (11)(12)(13)(14)(15)(16)(17)(18)22). Thus at present it is not possible to design directly zinc finger domains that specifically bind any given 3-bp DNA subsite.…”

“…Computer modeling of a helix binding to 5Ј-ACA-3Ј (SPA-D-LTR; Fig. 3e), based on the coordinates of finger 1 (QSG-S-LTR) of a Zif268 variant bound to 5Ј-GCA-3Ј (18), suggested that Arg 6 could easily adopt a configuration that allowed it to make a cross-strand hydrogen bond to O-4 of a thymine base-paired to the 5Ј-adenine (Fig. 7, …”

“…Direct interaction of the helix with all three bases of the DNA subsite has not been observed. Typically, only 1 or 2 bases of the 3-bp subsite are specified by residues in the ␣-helix (11)(12)(13)(14)(15)(16)(17)(18)22). Although these studies have revealed a variety of direct interactions between residues at helical positions Ϫ1 and 3 and the 3Ј-and middle nucleotides, respectively, the only direct interaction observed with an amino acid residue in position 6 of the ␣-helix and the 5Ј-nucleotide is an Arg 6 or Lys 6 to 5Ј-guanine.…”

“…In contrast to recognition of a 5Ј-guanine where a direct base contact is achieved by Arg or Lys in position 6 of the helix, no direct interaction has been observed in protein-DNA complexes for any other nucleotide in the 5Ј-position (11)(12)(13)(14)(15)(16)(17)(18). Selection of domains against finger-2 subsites of the type 5Ј-GNN-3Ј had previously generated domains containing only Arg 6 which directly contacts the 5Ј-guanine (7).…”

(Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a review

“…Borges and Collins 112 described that buffers, such as tricine (pH 8.0, 20 mmol l À1 ), affect the high-performance liquid chromatography (HPLC) stability and performance of study have themselves high complexing capabilities and buffer interference is thought to be simply nonexistent. These studies involve proteins with heme groups, [134][135][136] Zn-nger motifs, [137][138][139] metalloproteins 116,133,[140][141][142] and/or other complexing agents in solution. 81,[143][144][145] In fact, the concentrations of the compounds used, and most importantly the ratio of the buffer concentration to the complexing compound concentration in the medium, are within values that support the idea that no interference of the buffer occurs.…”