PDBsum entry 1jkp

DNA binding protein/DNA

PDB id: 1jkp

Contents

Protein chain

47 a.a. *

DNA/RNA

Waters ×4

* Residue conservation analysis

PDB id:

1jkp

Name:

DNA binding protein/DNA

Title:

Testing the water-mediated hin recombinase DNA recognition by systematic mutations

Structure:

5'-d( Tp Gp Tp Tp Tp Tp Tp Gp Ap Gp Ap Ap Gp A)-3'. Chain: a. Engineered: yes. 5'-d( Ap Tp Cp Tp Tp Cp Tp Cp Ap Ap Ap Ap Ap C)-3'. Chain: b. Engineered: yes. DNA-invertase hin. Chain: c. Fragment: residues 139 to 190.

Source:

Synthetic: yes. Other_details: synthetic peptide

Biol. unit:

Hexamer (from PQS)

Resolution:

2.80Å R-factor: 0.248 R-free: 0.328

Authors:

T.K.Chiu,C.Sohn,R.C.Johnson,R.E.Dickerson

Key ref:

T.K.Chiu et al. (2002). Testing water-mediated DNA recognition by the Hin recombinase. EMBO J, 21, 801-814. PubMed id: 11847127 DOI: 10.1093/emboj/21.4.801

Date:

13-Jul-01 Release date: 22-Feb-02

Protein chain

P03013  (HIN_SALTY) -  DNA-invertase hin from Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)

Seq: 190 a.a.

Struc: 47 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

T-G-T-T-T-T-T-G-A-G-A-A-G-A 14 bases

A-T-C-T-T-C-T-C-A-A-A-A-A-C 14 bases

DOI no: 10.1093/emboj/21.4.801

EMBO J 21:801-814 (2002)

PubMed id: 11847127

Testing water-mediated DNA recognition by the Hin recombinase.

T.K.Chiu, C.Sohn, R.E.Dickerson, R.C.Johnson.

ABSTRACT

The Hin recombinase specifically recognizes its DNA-binding site by means of both major and minor groove interactions. A previous X-ray structure, together with new structures of the Hin DNA-binding domain bound to a recombination half-site that were solved as part of the present study, have revealed that two ordered water molecules are present within the major groove interface. In this report, we test the importance of these waters directly by X-ray crystal structure analysis of complexes with four mutant DNA sequences. These structures, combined with their Hin-binding properties, provide strong support for the critical importance of one of the intermediate waters. A lesser but demonstrable role is ascribed to the second water molecule. The mutant structures also illustrate the prominent roles of thymine methyls both in stabilizing intermediate waters and in interfering with water or amino acid side chain interactions with DNA.

Selected figure(s)

Figure 2.
Figure 2 Details of Hin−DNA interactions within the major groove of Br18. (A) Stereo view of major groove interactions. Ser174 and the side chain of Arg178 within helix 3 are depicted in ball-and-stick representation. The methyl of T22 is rendered in space filling to illustrate its van der Waals interaction with W1. (B) Simulated-anneal F[o] - F[c] omit map of Br18 (contoured at 3.0 ), with the two interface water residues, Ser174 and Arg178, deleted from the final model during refinement from 5000K to 300K. (C) Ladder diagram of contacts within the major groove. D and A represent H-bond donors and acceptors: for adenine, A = N7 and D = N6; for thymine, A = O4; for guanine, A = N7 and O6; and for cytosine, D = N4. Bond distances given are for Br18. Lower case letters signify that the atoms are >3.5 Å away from either water or protein atoms.

Figure 5.
Figure 5 Hin−DNA interactions in models of T11A and A10T. (A) Energy-minimized model of T11A. The T21 and T22 methyl groups are rendered as van der Waals spheres. The T21-methyl occludes W2 but may stabilize W1. (B) Substitution of a T:A base pair at position 10 in mutant A10T results in a clash with the Ser174 side chain. The van der Waals surfaces of T10−C5A and Ser174-O are rendered. Substitution of a C:G base pair in mutant A10C also results in a clash with the Ser174 side chain (not shown). Note that saturable binding was never achieved with A10T or A10C, even at native Hin or Hin-DBD concentrations exceeding 1 M. Models were generated by visually fitting the substituted base pair into the Br18 structure, and for T11A, energy minimized using CNS (Brünger et al., 1998).

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2002, 21, 801-814) copyright 2002.

Figures were selected by an automated process.