Catalysis-associated Conformational Changes Revealed by Human CD38 Complexed with a Non-hydrolyzable Substrate Analog

Abstract

Cyclic ADP-ribose (cADPR) is a calcium mobilization messenger important for mediating a wide range of physiological functions. The endogenous levels of cADPR in mammalian tissues are primarily controlled by CD38, a multifunctional enzyme capable of both synthesizing and hydrolyzing cADPR. In this study, a novel non-hydrolyzable analog of cADPR, N1-cIDPR (N1-cyclic inosine diphosphate ribose), was utilized to elucidate the structural determinants involved in the hydrolysis of cADPR. N1-cIDPR inhibits CD38-catalyzed cADPR hydrolysis with an IC 50 of 0.26 mM. N1-cIDPR forms a complex with CD38 or its inactive mutant in which the catalytic residue Glu-226 is mutated. Both complexes have been determined by x-ray crystallography at 1.7 and 1.76 Å resolution, respectively. The results show that N1-cIDPR forms two hydrogen bonds (2.61 and 2.64 Å ) with Glu-226, confirming our previously proposed model for cADPR catalysis. Structural analyses reveal that both the enzyme and substrate cADPR undergo catalysis-associated conformational changes. From the enzyme side, residues Glu-146, Asp-147, and Trp-125 work collaboratively to facilitate the formation of the Michaelis complex. From the substrate side, cADPR is found to change its conformation to fit into the active site until it reaches the catalytic residue. The binary CD38-cADPR model described here represents the most detailed description of the CD38-catalyzed hydrolysis of cADPR at atomic resolution. Our structural model should provide insights into the design of effective cADPR analogs. Cyclic ADP-ribose (cADPR)3 is a novel cyclic nucleotide derived from NAD. It is metabolized by a family of proteins called ADP-ribosyl cyclases (1, 2). This cyclic nucleotide features a head-to-tail type of glycosidic linkage (N1-C1Ј) between N1 of its adenine and the anomeric carbon (C1Ј) of the terminal ribose (3, 4). Results obtained in the past decade have established the second messenger role of cADPR in mobilizing calcium stores in various cell types (reviewed in Refs. 5, 6). Its calcium signaling function has since been found to be more versatile and has additionally been shown to modulate calcium influx across the plasma membrane (7, 8) as well as regulate calcium homeostasis within the nucleus (9, 10).Human CD38 is a type II transmembrane glycoprotein containing a small N-terminal tail, a single transmembrane helix, and a large extra-membranous portion that possesses ADPribosyl cyclase activity. As a member of the cyclase family, CD38 not only can synthesize cADPR from NAD but also can hydrolyze NAD and cADPR to produce ADP-ribose (2,(11)(12)(13)(14). At acidic conditions, CD38 can also catalyze the formation and hydrolysis of NAADP, another calcium mobilization messenger (15,16). Although the mechanism of how these various activities of CD38 are regulated inside cells remains to be determined, we have previously identified residue Glu-146 as being critically important for controlling the relative synthesizing and hydrolyzing activities (16,17). The enzymatic portion of human ...

Structural Studies of Intermediates along the Cyclization Pathway of Aplysia ADP-Ribosyl Cyclase

“…This observation was also seen in the structures of human CD38, the homolog of Aplysia cyclase. [18][19][20] The results represent the first time ever that a fully active Aplysia cyclase has been observed with an NAD + molecule, its natural substrate, or an NGD + molecule, a close analog of NAD + , bound at the active site. The results also validate our approach of selecting crystals with multiple cyclase molecules in the asymmetric unit for analysis.…”

“…14 To decipher details of the cyclization reaction by Aplysia cyclase and to understand the structural differences conferring its homolog human CD38 to be more of an NAD + hydrolase than cyclase (i.e., converting NAD + to a linear ADPR instead of the cyclic product, cADPR), we have performed extensive kinetic crystallographic studies to attempt to arrest the reaction at specific steps. 12,13,[15][16][17][18][19][20][21] In most cases, the crystals of both proteins contained two molecules per asymmetric unit, and even though different states of the reaction intermediate can be captured in the two molecules, it could not provide us with a comprehensive picture about the progress of the reaction. Interestingly, Aplysia cyclase and human CD38 have the ability to crystallize in different crystal forms, some of which contain multiple subunits of the protein in the asymmetric unit.…”

Evolution and Function of the ADP Ribosyl Cyclase/CD38 Gene Family in Physiology and Pathology

“…More recently, the crystal structure of the human CD38 enzymatic domain complexed with cADPR and with its analog cyclic GDP-ribose and NGD at different resolutions indicated that the binding of cADPR (or cGDPR) to the active site induces significant structural rearrangements in the dipeptide Glu146-Asp147 (217). This provides direct evidence of a conformational change at the active site during catalysis.…”

Acetylcholine-Induced M/KCNQ Current Inhibition is Regulated by CD38 and A-Kinase Anchoring Protein in Neuroblastoma Cells

“…Furthermore, this is the first demonstration that CD38 is functionally involved in mAChR downstream signaling to modulate electrical membrane properties of neuronal tumor cells. When we examine with a catalytically dead mutant of CD38 such as an amino acid substitution from glutamic acid 226 to glycine (Munshi et al, 2000;Liu et al, 2007), our hypothesis would be strengthened.…”