Fluorometric studies of ligand-induced conformational changes of CD38.
The lymphoid surface antigen CD38 is a NAD(+)-glycohydrolase that also catalyzes the transformation of NAD(+) into cyclic ADP-ribose, a calcium mobilizing second messenger. In addition, ligation of CD38 by antibodies triggers signaling in lymphoid cells. Since the cytoplasmic tail of CD38 is dispensable for this latter property, we have previously proposed that CD38-mediated receptor signal transduction might be regulated by its conformational state. We have now examined the molecular changes of this protein during its interaction with NAD(+) by measuring the intrinsic fluorescence of CD38. We have shown that addition of the substrate produced a dramatic decrease in the fluorescence of the catalytically active recombinant soluble ectodomain of murine CD38. Analysis of this event revealed that the catalytic cycle involves a state of the enzyme that is characterized by a low fluorescence which, upon substrate turnover, reverts to the initial high intrinsic fluorescence level. In contrast, non-hydrolyzable substrates trap CD38 in its altered low fluorescence state. Studies with the hydrophilic quencher potassium iodide revealed that the tryptophan residues that are mainly involved in the observed changes in fluorescence, are remote from the active site. Similar data were also obtained with human CD38, indicating that studies of intrinsic fluorescence will be useful in monitoring the transconformation of CD38 from different species. Together, these data demonstrate that CD38 undergoes a reversible conformational change after substrate binding, and suggest a mechanism by which this change could alter interactions with different cell-surface partners.
INSERM U410, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France.
Jean-Jacques
JJ
Lacapère
Geneviève
G
Boulla
Frances E
FE
Lund
Julie
J
Primack
Norman
N
Oppenheimer
Francis
F
Schuber
Philippe
P
Deterre
D036541:ADP-ribosyl Cyclase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D051997:ADP-ribosyl Cyclase 1; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins; D051379:Mice; D008958:Models, Molecular; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D009536:Niacinamide / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D015703:Antigens, CD; D008024:Ligands; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D014364:Tryptophan; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1