Secondary and tertiary structural changes in γδ resolvase: Comparison of the wild‐type enzyme, the I11OR mutant, and the C‐terminal DNA binding domain in solution

Abstract

y6Resolvase is a site-specific DNA recombinase (MI 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I1 1OR resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I1 1OR mutant contains a folded catalytic core of -60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the IllOR resolvase. No increase in the helicity of the linker region of IllOR resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.Keywords: DNA; folding; interaction; NMR; protein; recombination; resolvase; structure y6 Resolvase (MI 20.5 kDa) is a site-specific DNA recombinase in Escherichia coli that shares homology with a family of resolvases and invertases in a variety of bacteria (Grindley & Reed, 1985). The structure of resolvase can be divided into three distinct regions, consisting of a catalytic domain (residues 1-120), a DNA binding domain (residues 149-183), and a linker region between the two globular domains. Limited proteolysis of 715 resolvase yields a C-terminal 43-residue fragment (5 kDa) with DNA binding activity and an N-terminal 140-residue fragment (15.5 kDa) with oligomerization and catalytic functions. The structure of the catalytic domain has been determined by X-ray crystallography

Serine Resolvases

“…The positions of the activating mutations map to the dimer interface region: helix E, the turn leading into it, and the surfaces that it packs against. It had already been noticed when comparing multiple structures that the WT dimer is quite flexible, and nuclear magnetic resonance data showed that helix E is not integral to the folding of the catalytic domain (43, 46, 47). Thus, it seemed likely that activation might involve a rearrangement of helix E relative to the rest of the catalytic domain.…”

Forcing Thermodynamically Unfolded Proteins to Fold

“…Examples of folding being driven by ligand binding include proteins with disordered domains that are induced to fold on binding DNA (1,23,24), the case of the (unstructured) prodomain of subtilisin being induced to fold when complexed with subtilisin (25,26), and several cases of preferential anion and/or cation binding to the native state of some proteins (23,27), shifting the equilibrium in favor of the folded state. According to the induced-fit model, ligand binding provides the driving forces for folding and the ligand serves as a template for the disordered domain to adopt its final conformation (1,28).…”

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C‐terminal domain

“…Cells were observed using an Olympus BX60 microscope equipped with a UPlan contrast objective (100 × magnification), and images were captured using a SenSys cooled CCD camera (Photometrics). Images were processed using V for Windows (Digital Optics) O. Sensitivity-enhanced, two-dimensional heteronuclear, single-quantum coherence (HSQC) spectra were acquired essentially as described previously (Kay et al, 1992;Pan et al, 1997) using flipback pulses and gradients for water suppression; the INEPT delay was 2.38 ms. NMR data were processed using Felix95 (MSI) and analysed using XEASY (Bartels et al, 1995).…”

“…NMR studies NMR studies of MinE 31-88 were performed at 25ЊC on a Varian INOVA 600 MHz spectrometer using samples of approximately 3 mM [U-15 N] MinE 31-88 dissolved in NMR buffer containing 7.5% D 2 O. Sensitivity-enhanced, two-dimensional heteronuclear, single-quantum coherence (HSQC) spectra were acquired essentially as described previously (Kay et al, 1992;Pan et al, 1997) using flipback pulses and gradients for water suppression; the INEPT delay was 2.38 ms. NMR data were processed using Felix95 (MSI) and analysed using XEASY (Bartels et al, 1995).…”