Structural basis for ligand binding to an enzyme by a conformational selection pathway

Abstract

Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the ratelimiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-Å X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic sidechains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.enzymatic catalysis | ligand binding | structural biology | adenylate kinase S ubstantial proportions of cellular processes depend on chemical reactions that in aqueous solution often are several orders of magnitude too slow to support biological life (1). This difference between "chemical" and "biological" time scales is bridged by acceleration of the rates of chemical reactions by enzymes (2). The catalytic power of an enzyme depends on a significant reduction of the free energy barrier for the chemical reaction (2). Several factors collectively contribute to an enzyme's efficiency as catalysts, including balanced substrate-binding affinity to ensure selectivity but at the same time avoid kinetic traps, optimal alignment of substrates, activation of a substrate's functional groups, and dehydration of active sites. All of these factors to some degree depend on conformational dynamics of the enzyme (3), where dynamics is defined as the time-dependent displacement of atomic coordinates. Structural excursions from enzymatic ground states to high-energy states have been observed with NMR spectroscopy, and in a few cases, were shown to be rate limiting for catalytic turnover (3-7). Furthermore, it has been proposed that the conformational dynamics required for substrate bindin...

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“…However, there is no justification that changes in the equilibrium constant can necessarily lead to an equally measureable effect on catalysis. Whilst our results agree with Kovermann et al [32] who found that an arrested closed conformation reduced catalytic activity, we can't fully conclude that there is a causal relationship. Aviram et al [33] have shown that there is a relationship between the domain opening and closing times that can measurably modulate enzymatic activity, however, this doesn't extend to the open and closed equilibrium.…”

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