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. 2011 Jan 28;405(4):1070-8.
doi: 10.1016/j.jmb.2010.11.044. Epub 2010 Dec 8.

Dynamic conformations of the CD38-mediated NAD cyclization captured in a single crystal

HongMin Zhang  1 , Richard Graeff, Zhe Chen, Liangren Zhang, Lihe Zhang, Honcheung Lee, Quan Hao
Affiliations

Dynamic conformations of the CD38-mediated NAD cyclization captured in a single crystal

HongMin Zhang et al. J Mol Biol. .

Abstract

The extracellular domain of human CD38 is a multifunctional enzyme involved in the metabolism of two Ca(2+) messengers: cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate. When NAD is used as substrate, CD38 predominantly hydrolyzes it to ADP-ribose, with a trace amount of cyclic ADP-ribose produced through cyclization of the substrate. However, mutation of a key residue at the active site, E146, inhibits the hydrolysis activity of CD38 but greatly increases its cyclization activity. To understand the role of the residue E146 in the catalytic process, we determined the crystal structure of the E146A mutant protein with a substrate analogue, arabinosyl-2'-fluoro-deoxy-nicotinamide adenine dinucleotide. The structure captured the enzymatic reaction intermediates in six different conformations in a crystallographic asymmetric unit. The structural results indicate a folding-back process for the adenine ring of the substrate and provide the first multiple snapshots of the process. Our approach of utilizing multiple molecules in the crystallographic asymmetric unit should be generally applicable for capturing the dynamic nature of enzymatic catalysis.

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Figures

Figure 1
Figure 1
Schematic diagram of the CD38-catalyzed reactions of NAD and ara-2′F-NAD. (A) Hydrolysis and cyclization of NAD catalyzed by human CD38. Human CD38 catalyzes the cleavage of the nicotinamide group from the northern ribose of NAD. In the hydrolysis reaction, a water molecule attacks the newly formed C-1′ atom producing linear product ADPR. In the cyclization reaction, the N1 atom of the adenine ring attacks the C-1′ atom producing cyclic product cADPR. (B) Hydrolysis of ara-2′F-NAD. The nicotinamide group is cleaved from the northern ribose and the newly generated C-1′ atom forms a covalent bond with the enzyme.
Figure 2
Figure 2
Overall structure of the E146A mutant. (A) The six molecules (labeled A-F) present in the asymmetric unit of the E146A crystals. Each protein molecule is shown as ribbons and contains one covalent bonded ara-2′ F-NAD that is shown as ball-and-stick models. (B) The Superposition of E146A molecules with wild type CD38 (PDB code 1YH3). Wild type CD38 is shown as red ribbons. Conformational variations are seen at the N-terminus, at a middle loop (G245–S250) and at the C-terminus. (C) The superposition of the six molecules in the asymmetric unit. The molecules are shown as Cα traces with molecules A and D colored in green and blue, respectively. The C domains of these molecules are well superimposed while the helices in the N domains show large variations indicated by the black arrows.
Figure 3
Figure 3
Stereo view of the superimposed wild type CD38 and E146A mutant at the active site. Wild type CD38 (colored in grey) in complex with ara-2′F-ADPR (PDB code 3I9M) is superimposed onto the E146A mutant (colored in cyan). Residues in the active site including E226, T221, W189, W125, E146/A146, H133 and D147 are shown as ball-and-stick models. Ara-2′F-ADPR in the E146A mutant structure is also shown as ball-and-stick models with yellow carbons. In wild type CD38, E146 forms hydrogen bonds with W125 and H133. In the E146A mutant, H133 swings away from the active site and forms a hydrogen bond with D147 in all six conformations.
Figure 4
Figure 4
Conformational variations of ara-2′F-ADPR in the active site of the E146A mutant. The protein is shown as cyan ribbons while ara-2′F-ADPR is shown as ball-and-stick models. Stereo view of ara-2′F-ADPR at the active site in N7 conformation (A) and in N1 conformation (B). The Fo-Fc map at 2.0σ is shown as grey mesh. (C) Stereo view of the active site of E146A mutant with ara-2′F-ADPR in the six molecules superimposed. Residues in the active site including W189, S193, E226, F222, T221, W125, S126 and R127 are also shown as ball-and-stick models with cyan carbons. The C-1′ atom is about 1.6Ǻ away from the OE2 oxygen of residue E226. The diphosphate part of ara-2′F-ADPR forms hydrogen bonds with main chain nitrogen atoms of F222 and T221 and side chains of S126 and R127. The adenine ring of ara-2′F-ADPR stacks with the side chain of W189 through hydrophobic interactions. (D) Ara-2′F-ADPR in molecule A, B and C. (E) Ara-2′F-ADPR in molecule D, E and F. The northern ribose and diphosphate part of ara-2′F-ADPR are well superimposed and the adenine rings are in different orientations as shown in (D) and (E). The black arrows indicate the movements of the adenine ring towards the northern ribose. (F) Comparison of conformations: the bound ara-2′F-ADPR in molecule A of the first group and that in molecule D of the second group. The adenine rings of molecules A and D are roughly related by a 180-degree rotation through the N6 atom of adenine and the C1′ atom of southern ribose as indicated by a red dash line.
Figure 5
Figure 5
A local view of two groups of ara-2′F-ADPR in the active site of the E146A mutant with a modeled E146. The wild type CD38 was superimposed onto the E146A mutant and only residues at the active site were shown to make the figure clear. Both ara-2′F-ADPR and residues in the active sites (with grey carbons) including E226, E146, W189 and D155 are shown as ball-and-stick models. In the N7 conformation (A), residues D155 and E146 form hydrogen bonds with the N1 and N6 atoms of the adenine ring of ara-2′F-ADPR while in the N1 conformation (B), they form hydrogen bonds with the N1, N6 and N7 atoms of the adenine ring.
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