PDBsum entry 2fr4

Immune system/DNA

PDB id: 2fr4

Contents

Protein chains

212 a.a. *

214 a.a. *

DNA/RNA

Ligands

PG4

Waters ×400

* Residue conservation analysis

PDB id:

2fr4

Name:

Immune system/DNA

Title:

Structure of fab DNA-1 complexed with a stem-loop DNA ligand

Structure:

5'-d( Cp Tp Gp Cp Cp Tp Tp Cp Ap G)-3'. Chain: m, n. Engineered: yes. Antibody light chain fab. Chain: l, a. Fragment: antigen-binding fragment. Engineered: yes. Antibody heavy chain fab. Chain: h, b.

Source:

Synthetic: yes. Other_details: single-stranded DNA oligonucleotide synthesized by idt. Mus musculus. House mouse. Organism_taxid: 10090. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Resolution:

1.95Å R-factor: 0.210 R-free: 0.256

Authors:

J.J.Tanner,Z.Ou

Key ref:

Z.Ou et al. (2007). Impact of DNA Hairpin Folding Energetics on Antibody-ssDNA Association. J Mol Biol, 374, 1029-1040. PubMed id: 18028946 DOI: 10.1016/j.jmb.2007.09.084

Date:

18-Jan-06 Release date: 09-Jan-07

Protein chains

No UniProt id for this chain

Struc: 212 a.a.

Protein chains

No UniProt id for this chain

Struc: 214 a.a.

DNA/RNA chains

C-T-G-C-C-T-T-C-A-G 10 bases

C-T-G-C-C-T-T-C-A-G 10 bases

DOI no: 10.1016/j.jmb.2007.09.084

J Mol Biol 374:1029-1040 (2007)

PubMed id: 18028946

Impact of DNA Hairpin Folding Energetics on Antibody-ssDNA Association.

Z.Ou, C.A.Bottoms, M.T.Henzl, J.J.Tanner.

ABSTRACT

Deposition of anti-DNA antibodies in the kidney contributes to the pathogenesis of the autoimmune disease, systemic lupus erythematosus. Antibodies that bind to hairpin-forming DNA ligands may be particularly prone to deposition. Here we report the first structure of a Fab complexed with hairpin-forming DNA. The ligand used for co-crystallization is 5'-d [CTG(CCTT)CAG]-3', which has a predicted hairpin structure consisting of a four-nucleotide loop (CCTT) and a stem of three base-pairs. The 1.95 A resolution crystal structure of Fab DNA-1 complexed with this ligand shows that the conformation of the bound ligand differs radically from the predicted hairpin conformation. The three base-pairs in the stem are absent in the bound form. The protein binds to the last six nucleotides at the 3' end of the ligand. These nucleotides form a loop (TTCA) closed by a G:C base-pair in the bound state. Stacking of aromatic side-chains against DNA bases is the dominant interaction in the complex. Interactions with the DNA backbone are conspicuously absent. Thermodynamics of binding are examined using isothermal titration calorimetry. The apparent dissociation constant is 4 muM, and binding is enthalpically favorable and entropically unfavorable. Increasing the number of base-pairs in the DNA stem from three to six decreases binding affinity. These data suggest a conformational selection binding mechanism in which the Fab binds preferentially to the unstructured state of the ligand. In this interpretation, the ligand binding and ligand folding equilibria are coupled, with lower hairpin stability leading to greater effective binding affinity. Thus, pre-organization of the DNA loop into the preferred binding conformation does not play a major role in complexation. Rather, it is argued that the stem of the hairpin serves to reduce the degrees of freedom in the free DNA ligand, thereby limiting the entropic cost attendant to complexation with the Fab.

Selected figure(s)

Figure 1.
Figure 1. Secondary structure diagrams for the DNA ligands used in this study. The three predicted hairpin conformations for LIG1-17 are shown in (a)–(c). The predicted hairpin conformation of LIG5-14 is shown in (d). The conformation of LIG5-14 bound to Fab DNA-1 is depicted in (e). The black and gray circles denote G:C and T:A base-pairs, respectively. The open circle represents a G:T wobble base-pair.

Figure 5.
Figure 5. Stereo view of one of the Fabs interacting with nucleotides 9–14. Dotted lines indicated hydrogen bonds. DNA is colored green. Light chain residues are colored magenta and appear on the right hand side of the Figure. Heavy chain residues are colored cyan and appear on the left hand side of the Figure. Compared to Figure 4, the orientation is rotated by about 180° around the vertical axis.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 374, 1029-1040) copyright 2007.

Figures were selected by the author.