Experimental Validation of the ALLNOX Program for Studying Protein-Nucleic Acid Complexes

Abstract

Measurement of distances between spectroscopic labels (e.g., spin labels, fluorophores) attached to specific sites of biomolecules is an important method for studying biomolecular complexes. ALLNOX (Addition of Labels and Linkers) has been developed as a program to model interlabel distances based on an input macromolecule structure. Here, we report validation of ALLNOX using measured distances between nitroxide spin labels attached to specific sites of a protein-DNA complex. The results demonstrate that ALLNOX predicts average interspin distances that matched with values measured with pairs of labels attached at the protein and/or DNA. This establishes a solid foundation for using spin labeling in conjunction with ALLNOX to investigate complexes without high-resolution structures. With its high degree of flexibility for the label or the target biomolecule, ALLNOX provides a useful tool for investigating the structure-function relationship in a large variety of biological molecules.

Figure 1.

Constructs for spin-labeling studies on GATA3 protein/DNA complex. (A) Chemical structure of the R5 label on DNA. (B) Chemical structure of the R5p label on protein, with the “label” shown in red, “linker” in blue, and “target” in black. (C) A crystal structure (4HCA.pdb) with the “double-finger” fragment of GATA3 bound to a DNA with palindromic GATA sites. The DNA is shown as a stick, the protein is shown as a ribbon with the domains colored, and the two zinc ions are shown in gray. (D) Schematic of the doublefinger GATA3 DNA-binding domain fragment (i.e., “DF”). The two zinc-fingers and the adjacent basic regions are color-coded as in panel (C). The detailed protein sequence is shown in Figure S1. (E) pGATA DNA duplex. The “GATA” motifs are shown in bold, and the pX15 and pY15 nucleotides used for R5 attachment are shown in red.

Figure 2.

Assessment of the interspin distance between a pair of R5p attached at T280 and I362 of the protein. (A) DEER measured background corrected echo evolution data on the DF/pGATA complex assembled with a protein/DNA ratio of 1:1.1. (B) Distance distribution profile computed from data shown in (A). The major population is shaded in yellow, and “*” indicates a minor population deemed “significant” on the basis of DEERconstruct analyses (see Supporting Information section S3). (C) ALLNOX generated structure of the spin-labeled DF/pGATA complex. The structure is depicted with the same scheme as described in Figure 1C. At the T280 and I362 site, one of the allowable R5p conformers is shown in stick form. (D) Histogram of ALLNOX predicted distance distribution.

Figure 3.

DEER data obtained in the DF/pGATA complex. (A) Pair of R5 labels at DNA pX15 and pY15 sites. To ensure all spin-labeled DNA was bound, the complex was assembled with a protein/DNA ratio of 1.1:1. (B) A R5p label at the protein and a R5 label at the DNA. Complexes were assembled with a protein/DNA ratio of 1:1. In each panel, on the left is the background correct echo evolution (black) with the fit trace (red) overlaid and on the right is the distance distribution profile with the major population shaded in yellow. “*” indicates a minor population deemed “significant” on the basis of DEERconstruct analyses (see Supporting Information section S3).

Figure 4.

GATA3 double-finger fragment binding to a DNA with a single GATA site. (A) sGATA DNA duplex. The “GATA” motif is in bold, and the R5 labeling sites are in red. (B) DEER result on I362–sX12 (top) and I362-sY18 (bottom). The complexes were assembled with a protein/DNA ratio of 1:1. In each data set, on the left is the background correct echo evolution (black) with the fit trace (red) overlaid and on the right is the distance distribution profile with the major population shaded in yellow. “*” indicates a minor population deemed “significant” on the basis of DEERconstruct analyses (see Supporting Information section S3). (C) ALLNOX generated spin-labeled complex structure. The structure is depicted with the same scheme as described in Figure 1C. One each of the allowable spin label conformers is shown at I362, sX12, and sY18. To mimic C-finger binding to one GATA site, the N-finger and the linker of GATA3 are not shown.