ADP-ribosyl cyclase is a multi-functional enzyme that catalyzes the formation of two Ca2+ signaling molecules, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). X-ray crystallography of three different crystal forms shows that it is a non-covalent dimer. Chemical cross-linking and dynamic light scattering were used in this study to determine if the cyclase is also a non-covalent dimer in solution. Treatment of the cyclase in dilute solution (0.05 mg/ml) with dimethylsuberimidate resulted in complete conversion to a species with molecular weight about twice that of the monomeric cyclase. Prolonged cross-linking of the cyclase at four times higher concentration produced also only the covalently linked dimers and no multimer formation was observed. The cross-linked dimer retained full enzymatic activity and readily catalyzed the formation of cADPR from NAD, NAADP from NADP, cyclic ADP-ribose phosphate from NADP, and cyclic GDP-ribose from nicotinamide guanine dinucleotide. Analysis of the autocorrelation functions obtained from dynamic light scattering measurements indicated the cyclase solution (2 mg/ml) was composed of a single molecular species and its diffusion coefficient was measured to be 7.4x10-7 cm2/s. Computer modeling using the crystallographic dimensions of the non-covalent cyclase dimer, a donut shaped molecule with a central cavity and overall dimensions of 7x6x3 nm, gave a value for the diffusion coefficient essentially the same as that measured. These results indicate the cyclase is a non-covalent dimer in solution. Copyright (C) 1998 Elsevier Science B.V.
|Original language||English (US)|
|Number of pages||9|
|Journal||Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology|
|State||Published - Nov 10 1998|
Bibliographical noteFunding Information:
This work was supported by grants from NIH, HD17484 to H.C.L and GM28093 to V.A.B. We thank Robert Aarhus for technical assistance.
Copyright 2007 Elsevier B.V., All rights reserved.
- ADP-ribosyl cyclase
- Crystallographic structure
- Cyclic ADP-ribose
- Dynamic laser-light scattering