Crystal Structure of a Soluble Form of Human CD73 with Ecto‐5′‐Nucleotidase Activity

Abstract

CD73 is a dimeric ecto-5'-nucleotidase that is expressed on the exterior side of the plasma membrane. CD73 has important regulatory functions in the extracellular metabolism of certain nucleoside monophosphates, in particular adenosine monophosphate, and has been linked to a number of pathological conditions such as cancer and myocardial ischaemia. Here, we present the crystal structure of a soluble form of human soluble CD73 (sCD73) at 2.2 Å resolution, a truncated form of CD73 that retains ecto-5'-nucleotidase activity. With this structure we obtained insight into the dimerisation of CD73, active site architecture, and a sense of secondary modifications of the protein. The crystal structure reveals a conserved loop that is directly involved in the dimer-dimer interaction showing that the two subunits of the dimer are not linked by disulfide bridges. Using biophotonic microarray imaging we were able to confirm glycosylation of the enzyme and show that the enzyme is decorated with a variety of oligosaccharide structures. The crystal structure of sCD73 will aid the design of inhibitors or activator molecules for the treatment of several diseases and prove useful in explaining the possible roles of single nucleotide polymorphisms in physiology and disease.

Streptococcal 5′-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion

“…Identified residues for phosphate binding (Asn-99 and Arg-392) and the predicted catalytic dyad residue His-100 were found to be critical for AMP hydrolysis. This suggests that the enzymatic mechanism is similar to what has been described for CD73 and E. coli 5Ј-nucleotidase (32,33).…”

“…In contrast, CD73 has been described as a zinc-dependent 5Ј-nucleosidase due to the fact that after stripping metal ions by EDTA treatment, Zn 2ϩ reconstitution resulted in the highest enzymatic activity when compared with other divalent cations. Furthermore, it was shown that CD73 is inactive with Mg 2ϩ as metal cofactor (32). Notably, the Zn 2ϩ concentration in human blood is ϳ6.2 mg/liter (ϳ0.1 mM) (42), which would allow optimal S5nA activity.…”

Nucleotides Acting at P2Y Receptors: Connecting Structure and Function

“…1A) and finally by 59-nucleotidase for the conversion to adenosine, which acts at its own set of four GPCRs. Recently, the structures of 59-nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase (CD39) were determined using X-ray crystallography (Heuts et al, 2012;Zimmermann et al, 2012). The structures of some of the other enzymes involved in processing purine receptor ligands, such as ecto-nucleotide pyrophosphatase/phosphodiesterase-1, have also been determined (Jansen et al, 2012).…”

5'-Nucleotidases, Nucleosides and their Distribution in the Brain: Pathological and Therapeutic Implications

“…Ecto-5'-nucleotidase is a dimeric extracellular glycoprotein with similar open and closed conformations to bacterial enzymes [121]. A crystal structure of the closed and open formations of human e'5NT have been obtained [174,175]. Structural control of the domain movement may be responsible for the selectivity of monophosphate nucleotides [176].…”