Electron Spin Resonance Shows Common Structural Features for Different Classes of EcoRI–DNA Complexes

Abstract

KeywordsEPR spectroscopy; DEER; DNA binding protein; structure elucidation; protein flexibility; noncognate complexIn this communication, we show that the EcoRI restriction endonuclease binds different classes of DNA sites in the same binding cleft. EcoRI generates widespread interest because it exhibits an extraordinary sequence selectivity to carry out its function of cleaving incoming foreign DNA without causing potentially lethal cleavage of cellular DNA. For example, EcoRI binds to its correct recognition site GAATTC up to 90,000-fold better than to miscognate sites that have one incorrect base pair. [1,2] The ∼650 specific sites in the E. coli genome are protected from cleavage by double-strand methylation. The ∼21,000 miscognate sites are not methylated, but are still cleaved by EcoRI with a second-order rate constant that is ∼10 9 -fold lower.[1,2] EcoRI forms only non-specific complexes with no cleavage at sites that differ from GAATTC by two or more base pairs. [1,2] In order to understand the source of such high specificity, it is necessary to determine how the structures of EcoRI complexes differ at specific, miscognate (5/6 bp match), and nonspecific (≤4/6 bp match) DNA sites. This effort is timely given the extensive genetic, biochemical and biophysical data on EcoRI.[1-9] Footprinting results [1] suggest that the three classes of complexes are "structurally" distinct, and thermodynamic profiles (ΔG°, ΔH°, ΔS°, ΔC°P) [3,4] suggest that the specific complex has more restricted conformationalvibrational mobility of the protein and DNA. There are crystal structures of the free protein, Figure 1 shows the structure of the EcoRI specific complex. [6,7] The protein contains a large, relatively rigid and structured globular "main" domain and a smaller "arm" region. The protein arms are invisible in the free protein[6] but become ordered and enfold the DNA in the specific complex, where they play a role in modulating specificity.[2,4] Mutations R131C, S180C, and K249C-S180C were chosen based on the crystal structure. [6,7] These sites are solvent accessible and therefore likely to spin label with minimal perturbation to protein structure. Residues R131 and S180 lie in the inner and outer arms, respectively. Residue K249 is in the main domain, which has very restricted movement [6] and acts as a reference point. Since EcoRI is a 62 kDa homodimer, single cysteine mutations provide two sites for spin labeling, and double mutations provide four sites.The proteins were spin labeled at the cysteines with the methanethiosulfonate spin label (MTSSL). There is an intrinsic cysteine at position 218, but it is buried, leading to <10% labeling even with a 100-fold molar excess of the spin label. The mutant proteins and their spin labeled derivatives catalyze DNA cleavage and have DNA binding affinities similar to that of wild type EcoRI, indicating that they are functionally active (Supporting Information).DEER experiments [11] were performed on spin labeled S180C specific and non-specific complexes, and on R131C and...

Thermodynamic and Structural Basis for Relaxation of Specificity in Protein–DNA Recognition

“…The case of EcoRI endonuclease and its promiscuous mutants shows that we cannot think of specificity change in terms of addition or subtraction of contact points in a recognition complex, but must anticipate the possibility of significant structural and thermodynamic differences between complexes. There is reason for hope that spectroscopic methods may be an efficient approach to elucidating the differences among the various adaptive states, as well as the roles in specificity for dynamic motions of protein and DNA 25; 72-76 .…”

“…Further, when the free enzyme binds to the specific DNA recognition site, two “arms” protruding from the main domain of the endonuclease undergo a disorder to order transition 24 so as to make critical contacts to bases and phosphates. The most probable position of the arms is similar in specific and EcoRI* complexes, yet the distribution of positions is broadened in the EcoRI* complex 25 , suggesting the possibility that the EcoRI* complex ensemble is more populated by conformers that lack contacts between the arms and DNA. Finally, the protein-DNA contacts that stabilize the specific complex persist in the transition state 2 ., such that binding energy is utilized efficiently at the catalytic step; thus, the complex with the GAATTC site has been called a “pre-transition state” complex 2 .…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…884 Interactions between the restriction endonuclease EcoR1 and DNA has also been studied using EPR. 885 The rigid spin-label cytosine analogue (40) has been synthesised and forms stable base pairs with guanine, and was used to study the motion within an oligonucleotides that can adopt two different hairpin conformations. 177 Raman spectroscopy has been used in a study with EM to investigate the global structure of DNA binding to the bacteriophage P22 terminase subunit.…”

Rotameric preferences of a protein spin label at edge‐strand β‐sheet sites

“…Side-chain motions dominate distance distributions between R1 pairs and make it challenging to extract C a AC a distances from such measurements. For example, we previously generated quantitative comparisons of the C a AC a distribution within a protein-DNA complex by comparison of distance distributions obtained by double electron electron resonance (DEER) 16 and molecular dynamics (MD) simulations; the C a AC a distribution from the simulation had a width six times narrower than the DEER experiment. 17 In an effort to account for R1 flexibility in distance measurements, various computational tools have been developed.…”