PDBsum entry 1pvq

Recombination/DNA

PDB id: 1pvq

Contents

Protein chains

323 a.a. *

DNA/RNA

Waters ×67

* Residue conservation analysis

PDB id:

1pvq

Name:

Recombination/DNA

Title:

Basis for a switch in substrate specificity: crystal structure of selected variant of cre site-specific recombinase, lnsgg bound to the engineered recognition site loxm7

Structure:

DNA 34-mer. Chain: c. Engineered: yes. Other_details: top strand of loxm7 engineered DNA substrate (loxp(c7/g28,t8/a27,a9/t26). DNA 34-mer. Chain: d. Engineered: yes. Other_details: bottom strand of loxm7 engineered DNA substrate

Source:

Synthetic: yes. Escherichia virus p1. Organism_taxid: 10678. Escherichia phage p1. Bacteriophage p1. Gene: cre. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Octamer (from PDB file)

Resolution:

2.75Å R-factor: 0.224 R-free: 0.281

Authors:

E.P.Baldwin,S.S.Martin,J.Abel,K.A.Gelato,H.Kim,P.G.Schultz, S.W.Santoro

Key ref:

E.P.Baldwin et al. (2003). A specificity switch in selected cre recombinase variants is mediated by macromolecular plasticity and water. Chem Biol, 10, 1085-1094. PubMed id: 14652076 DOI: 10.1016/j.chembiol.2003.10.015

Date:

28-Jun-03 Release date: 17-Feb-04

Protein chains

P06956  (RECR_BPP1) -  Recombinase cre from Escherichia phage P1

Seq: 343 a.a.

Struc: 323 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

DNA/RNA chains

A-T-A-A-C-T-C-T-A-T-A-T-A-A-T-G-T-A-T-G-C-T-A-T-A-T-A-G-A-G-T-T-A-T 34 bases

A-T-A-A-C-T-C-T-A-T-A-T-A-G-C-A-T-A-C-A-T-T-A-T-A-T-A-G-A-G-T-T-A-T 34 bases

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1016/j.chembiol.2003.10.015

Chem Biol 10:1085-1094 (2003)

PubMed id: 14652076

A specificity switch in selected cre recombinase variants is mediated by macromolecular plasticity and water.

E.P.Baldwin, S.S.Martin, J.Abel, K.A.Gelato, H.Kim, P.G.Schultz, S.W.Santoro.

ABSTRACT

The basis for the altered DNA specificities of two Cre recombinase variants, obtained by mutation and selection, was revealed by their cocrystal structures. The proteins share similar substitutions but differ in their preferences for the natural LoxP substrate and an engineered substrate that is inactive with wild-type Cre, LoxM7. One variant preferentially recombines LoxM7 and contacts the substituted bases through a hydrated network of novel interlocking protein-DNA contacts. The other variant recognizes both LoxP and LoxM7 utilizing the same DNA backbone contact but different base contacts, facilitated by an unexpected DNA shift. Assisted by water, novel interaction networks can arise from few protein substitutions, suggesting how new DNA binding specificities might evolve. The contributions of macromolecular plasticity and water networks in specific DNA recognition observed here present a challenge for predictive schemes.

Selected figure(s)

Figure 3.
Figure 3. Details of ALSHG/LoxM7 ComplexCre/LoxP (green sticks) was superimposed on ALSHG/LoxM7 (atom-colored balls and sticks) as described in Figure 2. The dashed lines represent potential hydrogen bonds in ALSHG/LoxM7 (black) and Cre/LoxP (yellow).(A) Specific contacts to bases C7 and T8. Residues 258–266 of helix J are rolled 7° and shifted 0.6 Å toward the DNA as a consequence of steric interactions between Leu258 and Ala175. This repositioning facilitates hydrogen bonding between Ser259 O^γ and C7 O^4 atoms. In addition, a network involving water molecules Sol67, Sol179, and Sol503 (B factors of 45, 52, and 50 Å^2, respectively) and the Ser257 O^γ atom, the Leu258 N atom, and the Ser259 N and O^γ atoms couples recognition of bases C7 and T8 and replaces the water bridge between Thr258 O^γ1 atom and the N^4 atom of base C8 in Cre/LoxP.(B) Coupled recognition of nucleotide T26, base A27, and the phosphate backbone via a tripartite hydrogen bond bridge. Base A27 is contacted by a hydrogen bond bridge mediated by Sol501 and Sol502 with the Ser259 carbonyl. His262 is rotated from the position of Glu262 in Cre/LoxP, which avoids a steric clash and forms a tight Van der Waals contact with the 5-methyl group of base T26. In addition, His262 forms a hydrogen bond bridge between Sol501 and the phosphate of nucleotide 26, connecting the T26 and A27 contacts.(C) Atomic level details of ALSHG/LoxM7 interactions. Symbols and distances are as described in Figure 1D.

Figure 4.
Figure 4. Structure of the Substituted Region of the LNSGG/LoxM7 ComplexFor comparison, Cre/LoxP (green sticks) or ALSHG/LoxM7 (purple sticks) are superimposed on LNSGG/LoxM7 (atom-colored balls and sticks), as described in Figure 2. Potential hydrogen bonds are denoted by dashed lines.(A) LNSGG/LoxM7 has contacts between the DNA backbone and base C7 but not bases A27 and T26. Helix J maintains a position similar to that in Cre/LoxP-G5. Ser259 forms a hydrogen bond with C7, and Asn258 is positioned to form hydrogen bond with the phosphate backbone at residue 24 (orange dashes). In addition, Sol49 and new solvents Sol501 and Sol505 (B factors of 61, 52, and 51 Å^2, respectively), form a hydrogen bond network that interconnects the Ser259 carbonyl with the phosphates of nucleotides 25 and 26. Sol49 and Sol84 occupy similar positions in Cre/LoxP-G5. Although Sol502 is still bound by A27, the increased length of the bridging contact with Sol501 (3.6 Å) indicates a weaker protein-DNA interaction.(B) Since helix J is not rotated as in ALSHG/LoxM7 (purple) and Sol501 is shifted toward Gly262, water molecules Sol501 and Sol502 are 1.2 Å farther apart (gray dashed lines) than in ALSHG/LoxM7 (cyan dashed lines), perhaps diminishing the strength of the contact. Note the correspondences of Sol49 and Sol505 in LNSGG and His262 in ALSHG. Sol84 is conserved in the Cre/LoxP-G5 and 1CRX structures.(C) Atomic level details of LNSGG/LoxM7 interactions. Symbols and distances are as described in Figure 1D. The gray stippled line indicates a weakened hydrogen bond with a contact distance that is greater than 3.5 Å.

The above figures are reprinted by permission from Cell Press: Chem Biol (2003, 10, 1085-1094) copyright 2003.

Figures were selected by the author.

Author's comment

This work demonstrates 1) that solvent molecules can arise as key players for achieving new DNA specicifity in directed evolution experiements (1pvp). Given the flexibility in hydrogen bonding and positioning compared to more constrained protein side-chains, perhaps solvent-mediated specificity networks arise with a higher frequency than side-chain networks; 2) that DNA flexibility can lead to difficult-to-predict protein-DNA interactions as exemplified by the differences in Ser259 interactions between LNSGG/LoxP and LNSGG/LoxM7 complex structures (1pvq vs 1pvr).