CD39, NTPDase 1, is attached to the plasma membrane by two transmembrane domains. Why?

Abstract

Since the identification of CD39 and other members of the e-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) family as the primary enzymes responsible for cell surface nucleotide hydrolysis, one of their most intriguing features has been their unusual topology. The active site lies in the large extracellular region, but instead of being anchored in the membrane by a single transmembrane domain or lipid link like other ectoenzymes, CD39 has two transmembrane domains, one at each end. In this review we discuss evidence that the structure and dynamics of the transmembrane helices are intricately connected to enzymatic function. Removal of either or both transmembrane domains or disruption of their native state by detergent solubilization reduces activity by 90%, indicating that native function requires both transmembrane domains to be present and in the membrane. Enzymatic and mutational analysis of the native and truncated forms has shown that the active site can exist in distinct functional states characterized by different total activities, substrate specificities, hydrolysis mechanisms, and intermediate ADP release during ATP hydrolysis, depending on the state of the transmembrane domains. Disulfide crosslinking of cysteines introduced within the transmembrane helices revealed that they interact within and between molecules, in particular near the extracellular domain, and that activity depends on their organization. Both helices exhibit a high degree of rotational mobility, and the ability to undergo dynamic motions is required for activity and regulated by substrate binding. Recent reports suggest that membrane composition can regulate NTPDase activity. We propose that mechanical bilayer properties, potentially elasticity, might regulate CD39 by altering the balance between stability and mobility of its transmembrane domains.

Figure 1

Cartoon of the structure of CD39. The region labeled PM represents the lipid bilayer of the plasma membrane. TM1 and TM2 are the N-terminal and the C-terminal transmembrane domains, respectively. The hourglass structures attached to TM1 and TM2 represent ACR1 and ACR5, respectively; they are part of the extracellular domain of CD39 (cylinder). The dynamic interactions between TM1 and TM2 in wild type (WT) CD39 may position ACR1 and ACR5 so that the configurations and mobility of the enzymatic active site are optimal and the specific activity is maximal. Removal of TM1 (ΔTM1), TM2 (ΔTM) or both transmembrane domains (ΔTM1 + ΔTM2) eliminates the interactions between TM1 and TM2 and would be expected to disrupt the alignment of ACR1 and ACR5 and of the enzymatic active site to bring about a loss of specific activity.