Cofactor Binding Modulates the Conformational Stabilities and Unfolding Patterns of NAD+-dependent DNA Ligases from Escherichia coli and Thermus scotoductus

Abstract

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD ؉ -dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD ؉ -DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD ؉ -dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.DNA ligases form a large family of evolutionarily related proteins that play important roles in a wide range of DNA transactions, including chromosomal DNA replication, DNA repair, and DNA recombination in all three kingdoms of life (1). Cofactor requirements divide the ligases into two subfamilies, the NAD ϩ -dependent DNA ligases and the ATP-dependent DNA ligases. Regardless of their energy source, they catalyze the sealing of 5Ј-phosphate and 3Ј-hydroxyl termini at nicks in duplex DNA by means of three distinct catalytic events (1). The first step involves activation of the ligase through the formation of a covalent adenylated intermediate by transfer of the adenyl group of NAD ϩ or ATP to the ⑀-NH 2 of a conserved lysine residue in the DNA ligase. In the second step the AMP moiety is transferred from the DNA ligase to the 5Ј-phosphate group at the single-strand break site, creating a new pyrophosphate bond. Finally, the phosphodiester bond formation is achieved upon an attack of the 3Ј-OH group of the DNA on the activated 5Ј-group with the concomitant release of AMP (1).At least one NAD ϩ -dependent DNA ligase (referred to as LigA) is found in every bacterial species (2). The bacterial LigA enzymes are of fairly uniform size (ϳ70 kDa) and display extensive amino acid sequence conservation throughout the entire protein (3-4). The atomic structures of the LigA enzymes from Bacillus stearothermophilus (N-terminal domain) (5) and Thermus filiformis (3) have been determined by x-ray crystallography. The catalytic core of the bacterial NAD ϩ -dependent DNA ligase con...

Characterization of a temperature-sensitive DNA ligase from Escherichia coli

“…Although the relationship of the amino acid mutation to enzymic activity or structure has not been directly demonstrated at this time, the molecular model suggests that the alteration of amino acid 15 from Leu to Phe may alter the packing involving a-helix A, which is close to the N-terminus. This bi-helix is implicated in the binding of NAD + to LigA (Georlette et al, 2003;Sriskanda & Shuman, 2002), supporting our proposal that the temperature sensitivity of LigA251 is a consequence of factors altering the rate of adenylation of the protein.…”

“…Thus, NAD + -DNA ligases have been suggested as possible targets for broad-spectrum antibacterial compounds (Georlette et al, 2003;Gong et al, 2004;Kaczmarek et al, 2001;Lee et al, 2000;Singleton et al, 1999;Sriskanda & Shuman, 2002). If substantial progress is to be made in the development of inhibitors that are specific to NAD + -DNA ligases, it is vital that in vivo models are utilized to test the efficacy of such compounds.…”

Human DNA Ligase IV and the Ligase IV/XRCC4 Complex: Analysis of Nick Ligation Fidelity

“…Consistent with the former, even at early stages of ligase IV/XRCC4 purification, only small quantities of labeled protein are detected upon incubation with Mg 2+ and [R-32 P]ATP; consistent with the latter, we have found the unadenylylated form of ligase IV (in both its free and XRCC4-complexed forms) to be less stable than the adenylylated form during extended periods of incubation at 37°C (data not shown). Studies with other DNA ligases have demonstrated the presence of a conformational change upon enzyme adenylylation (39)(40)(41)(42); in some cases, this was found to enhance thermostability (41,42). In light of the above, the inclusion of a Mg 2+ /ATP incubation step at an early phase of protein purification could conceivably enhance the yield of ligase IV.…”

Cold-Active Winter Rye Glucanases with Ice-Binding Capacity

“…Structural studies have revealed that these enzymes are generally highly fold flexible and therefore less rigid (Georlette et al, 2003(Georlette et al, , 2004Collins et al, 2005). Cold-active enzymes are also characterized by stacking of small amino acids around the catalytic residues (Feller et al, 1992;Davail et al, 1994).…”