Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases

Abstract

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 Å ), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.

The TB Structural Genomics Consortium: A decade of progress

“…To improve the overall success rate through effective salvaging pathways, the protein production facility has incorporated two emerging techniques; surface entropy reduction (SER) 24 and high-throughput ligand analysis 25 , into its production protocols. As proof of concept, six Mtb targets with poor crystallizabilities were selected for the SER process.…”

Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

“…[17]) for obtaining diffraction data by crystallization improvement. The structure of each protein for which we have collected data has been solved and is in the process of refinement (these structures will be reported elsewhere).…”

“…[17]. Briefly, individual purified proteins were diluted to 1–2 mg/ml in column buffer (CB; 50 mM potassium phosphate, pH 7.5, 1 mM MgCl 2 and 2 mM DTT), and 100 μg protein was adsorbed to multiple 50 μl aliquots of F3GA resin (Bio-RAD) in 2 ml spin-columns (Costar, Fisher Scientific).…”

“…If the right ligand is chosen, the effect on crystallization can be dramatic [17, 18]. In practice, the success rate of this approach is limited by the accuracy of ligand prediction for target proteins.…”

Affinity-Based Profiling of Dehydrogenase Subproteomes

“…But, the use of NAD(P) as a ligand, either as a scaffold for building a drug or as part of an affinity matrix, is not ideal because of its instability and poor bioavailability, which is why we developed the use of the catechol rhodanine ligand (9, 16, 17). Most recently, Kim et al used a Cibacron Blue F3GA dye affinity column to ligand-specifically elute and identify aldehyde dehydrogenases from Mycobacterium tuberculosis (18). These approaches demonstrate that dehydrogenase subproteomes can be purified and analyzed using affinity chromatography (19).…”