Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.
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 A), 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.
Kim
Chang-Yub
CY
Advanced Measurement Science Group (B-9), Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM, 87545, USA. cykim@lanl.gov
Webster
Cecelia
C
Roberts
Justin K M
JK
Moon
Jin Ho
JH
Alipio Lyon
Emily Z
EZ
Kim
Heungbok
H
Yu
Minmin
M
Hung
Li-Wei
LW
Terwilliger
Thomas C
TC
D000444:Aldehyde Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009705:Nucleosides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry
D001426:Bacterial Proteins; D007527:Isoenzymes; D024101:Mitochondrial Proteins; D009705:Nucleosides; D011994:Recombinant Proteins; C017842:cibacron blue-sepharose; D012685:Sepharose; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial