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

Abstract

As a novel approach to the structural and functional properties that give rise to extremely stringent sequence specificity in protein-DNA interactions, we have exploited “promiscuous” mutants of EcoRI endonuclease to study the detailed mechanism by which changes in a protein can relax specificity. The A138T promiscuous mutant protein binds more tightly to the cognate GAATTC site than does wild-type EcoRI, yet displays relaxed specificity deriving from tighter binding and faster cleavage at EcoRI* sites (one incorrect base pair). AAATTC EcoRI* sites are cleaved by A138T up to 170-fold faster than by wild type enzyme if the site is abutted by a 5’-purine-pyrimidine (5’-RY) motif. When wild-type protein binds to an EcoRI* site, it forms structurally adapted complexes with thermodynamic parameters of binding that differ markedly from those of specific complexes. By contrast, we show that A138T complexes with 5’-RY-flanked AAATTC sites are virtually indistinguishable from wild-type specific complexes with respect to the heat capacity change upon binding (ΔC°P), the change in excluded macromolecular volume upon association, and contacts to the phosphate backbone. While the preference for the 5’-RY motif implicates contacts to flanking bases as important for relaxed specificity, local effects are not sufficient to explain the large differences in ΔC°P and excluded volume, as these parameters report on global features of the complex. Our findings therefore support the view that specificity does not derive from the additive effects of individual interactions, but rather from a set of cooperative events that are uniquely associated with specific recognition.

Insights into copper coordination in the EcoRI–DNA complex by ESR spectroscopy

“…It carries out staggered cleavage of the DNA sequence 5′-GAATTC-3′ in both strands, generating single-strand overhangs. Cleavage rates are as much as 10 6 -fold lower at DNA sites that differ in one base-pair from the canonical GAATTC [2–5]. The enzyme is a symmetrical homodimer with two catalytic centres, in each of which Mg 2+ coordinates to carboxylate side chains of E111, D91, and the scissile phosphate GpAATTC [4,6].…”

Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems

“…to the conclusion that Δ w is small if salts in the middle of the Hofmeister series, [108] for example, NaCl or KCl, are used. Hence, Δ w will be small, and nearly all complexes of DNA with proteins have been modeled by Equation (3a) [2, 10, 13, 14, 46–49] …”

“…DNA is perhaps the best‐studied natural polyelectrolyte and in the preceding 50 years most work has been directed towards a detailed understanding of the interaction between DNA and proteins related to DNA repair proteins or transcription factors [2–11] . The thermodynamics of the binding of DNA or RNA to proteins has been shown to be dominated by charge–charge interactions, and the biological activity of natural polyelectrolytes such as DNA is intimately related to their highly charged molecular structures [2, 12–15] . Heparin provides another example of a natural polyelectrolyte with four charges per repeating unit that has been studied intensively during the last 30 years [16–19] …”

“…Inspired by earlier work on the interaction of DNA with various proteins, [2, 10, 13, 14, 46–49] we recently reconsidered the problem of counterion release by a series of thermodynamic studies related to the interaction of polyelectrolytes with proteins. First, the interaction of human serum albumin (HAS) with short‐chain poly(acrylic acid) in aqueous solution was studied by a combination of calorimetry and molecular dynamics simulations [50] .…”

Wechselwirkung von Polyelektrolyt‐Architekturen mit Proteinen und Biosystemen

“…Der wahrscheinlich am besten erforschte natürliche Polyelektrolyt ist die DNA, weshalb in den letzten 50 Jahren hauptsächlich auf ein detailliertes Verständnis der Interaktion zwischen DNA und Proteinen im Zusammenhang mit DNA‐Reparaturproteinen oder Transkriptionsfaktoren hingearbeitet wurde [2–11] . Dabei hat sich herausgestellt, dass die Thermodynamik der Bindung von DNA oder RNA an Proteine von der Ladungs‐Ladungs‐Wechselwirkung dominiert wird und dass die biologische Aktivität natürlicher Polyelektrolyte wie der DNA in enger Beziehung zu ihrer hochgeladenen Molekülstruktur steht [2, 12–15] . Heparin ist ein weiteres Beispiel eines natürlichen Polyelektrolyten mit vier Ladungen pro Wiederholungseinheit, der in den letzten 30 Jahren intensiv untersucht wurde [16–19] …”