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Adenylation-Dependent Conformation and Unfolding Pathways of the NAD⁺-Dependent DNA Ligase from the Thermophile Thermus scotoductus

Daphné Georlette ^* **, Vinciane Blaise ^*, Fabrice Bouillenne , Benjamin Damien , Sigridur H. Thorbjarnardóttir , Eric Depiereux , Charles Gerday ^*, Vladimir N. Uversky ^¶ || and Georges Feller ^*

^* Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium; Laboratoire d'Enzymologie et Centre d'Ingénierie des Protéines, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium; Unité de Biologie Moléculaire, Département de Biologie, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium; Laboratory of Molecular Genetics, Institute of Biology, University of Iceland, Grensasvegur 12, IS-108 Reykjavik, Iceland; ^¶ Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia; ^|| Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064; and ^** Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204

Correspondence: Address reprint requests to Georges Feller, Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium. Tel.: +32-4-366-33-43; Fax: +32-4-366-33-64; E-mail: gfeller{at}ulg.ac.be.

In the last few years, an increased attention has been focused on NAD⁺-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD⁺-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD⁺-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates.