PDBsum entry 1qqb

Gene regulation/DNA

PDB id: 1qqb

Contents

Protein chain

338 a.a. *

DNA/RNA

Ligands

HPA

Waters ×57

* Residue conservation analysis

PDB id:

1qqb

Name:

Gene regulation/DNA

Title:

Purine repressor mutant-hypoxanthine-palindromic operator complex

Structure:

5'-d( Tp Ap Cp Gp Cp Ap Ap Tp Cp Gp Ap Tp Tp Gp Cp Gp T)- 3'. Chain: m. Engineered: yes. Purine nucleotide synthesis repressor. Chain: a. Synonym: pura. Engineered: yes

Source:

Synthetic: yes. Escherichia coli. Organism_taxid: 562. Gene: purr. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.70Å R-factor: 0.165

Authors:

A.Glasfeld,A.N.Koehler,M.A.Schumacher,R.G.Brennan

Key ref:

A.Glasfeld et al. (1999). The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions. J Mol Biol, 291, 347-361. PubMed id: 10438625 DOI: 10.1006/jmbi.1999.2946

Date:

01-Jun-99 Release date: 09-Jun-99

Protein chain

P0ACP7  (PURR_ECOLI) -  HTH-type transcriptional repressor PurR from Escherichia coli (strain K12)

Seq: 341 a.a.

Struc: 338 a.a.*

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

DNA/RNA chain

T-A-C-G-C-A-A-T-C-G-A-T-T-G-C-G-T 17 bases

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1006/jmbi.1999.2946

J Mol Biol 291:347-361 (1999)

PubMed id: 10438625

The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions.

A.Glasfeld, A.N.Koehler, M.A.Schumacher, R.G.Brennan.

ABSTRACT

The interaction of the dimeric Escherichia coli purine repressor (PurR) with its cognate sequences leads to a 45 degrees to 50 degrees kink at a central CpG base step towards the major groove, as dyad-related leucine side-chains interdigitate between these bases from the minor groove. The resulting broadening of the minor groove increases the accessibility of the six central base-pairs towards minor groove interactions with residues from PurR. It has been shown that lysine 55 of PurR makes a direct contact with the adenine base (Ade8) directly 5' to the central CpG base-pair step in the high-affinity purF operator sequence. We have investigated the importance of this interaction in the specificity and affinity of wild-type PurR (WT) for its operators and we have studied a mutant of PurR in which Lys55 is replaced with alanine (K55A). Complexes of WT and K55A with duplex DNA containing pur operator sequences varied at position 8 were investigated crystallographically, and binding studies were performed using fluorescence anisotropy. The structures of the protein-DNA complexes reveal a relatively unperturbed global conformation regardless of the identity of the base-pair at position 8 or residue 55. In all structures the combination of higher resolution and a palindromic purF operator site allowed several new PurR.DNA interactions to be observed, including contacts by Thr15, Thr16 and His20. The side-chain of Lys55 makes productive, though varying, interactions with the adenine, thymine or cytosine base at position 8 that result in equilibrium dissociation constants of 2.6 nM, 10 nM and 35 nM, respectively. However, the bulk of the lysine side-chain apparently blocks high-affinity binding of operators with guanine at position 8 (Kd620 nM). Also, the high-affinity binding conformation appears blocked, as crystals of WT bound to DNA with guanine at position 8 could not be grown. In complexes containing K55A, the alanine side-chain is too far removed to engage in van der Waals interactions with the operator, and, with the loss of the general electrostatic interaction between the phosphate backbone and the ammonium group of lysine, K55A binds each operator weakly. However, the mutation leads to a swap of specificity of PurR for the base at position 8, with K55A exhibiting a twofold preference for guanine over adenine. In addition to defining the role of Lys55 in PurR minor groove binding, these studies provide structural insight into the minor groove binding specificities of other LacI/GalR family members that have either alanine (e.g. LacI, GalR, CcpA) or a basic residue (e.g. RafR, ScrR, RbtR) at the comparable position.

Selected figure(s)

Figure 3.
Figure 3. An overlay of the models of the DNA-bind- ing domain of WT PurR (blue) complexed to ApC (cyan) and the DNA-binding domain of the K55A mutant of PurR (red) complexed to ApC (pink). Lys55 of WT PurR is shown in green with its van der Waals surface. The image was prepared with MidasPlus soft- ware (Ferrin et al., 1988).

Figure 4.
Figure 4. Interactions made by Thr16 to the major groove of the palindromic pur operator. The side- chain hydroxyl group makes three potential hydrogen bonds to bases in the operator sequence, and the C g group potentially interacts with the C5 methyl group of Thy60.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1999, 291, 347-361) copyright 1999.

Figures were selected by an automated process.