Structural elements that regulate interactions between the extracellular and transmembrane domains of human nucleoside triphosphate diphosphohydrolase 3
The nucleoside triphosphate diphosphohydrol ases (NTPDases) are a family of constitutively expressed, endogenous nucleotidases, some of which regulate purinergic signaling by divalent cation-dependent hydrolysis of nucleotides acting as agonists at purinergic receptors. Due to the scarcity of specific inhibitors and genetically modified animals, the functions of most individual NTPDases are poorly understood and still under investigation. However, the NTPDases have been implicated in many biological and physiological processes, including secretion, cell adhesion, pain perception, cancer and malignant transformation, adenosine recycling, and platelet aggregation.
Membrane-bound NTPDase3 expressed on the cell surface has a large extracellular domain, a “linker region”, and a transmembrane domain. Evidence exists for interactions between the transmembrane domain and the active site lobes that govern the function of rat NTPDase1. However, the specific mechanism(s) regulating this cross-talk for NTPDases, as well as how the N- and C-terminal transmembrane helices in NTPDase3 interact within and between monomers to mediate oligomerization and modulation of enzymatic activity is still unknown.
The roles of the conserved proline residues of human NTPDase3, located in the “linker region” that connects the N- and C-terminal transmembrane helices with the extracellular active site, were examined by proline to alanine substitutions coupled with single cysteine substitutions strategically placed in the transmembrane domain to serve as cross-linking “sensors” of helical interactions. Mutation of several proline residues resulted in decreased nucleotidase activities and some “uncoupled” the effect of ATP binding on TMD movements. The data suggest a role for proline residues 53 and 481 in the linker region of human NTPDase3 for “coupling” nucleotide binding and hydrolysis at the enzyme active site to movements and/or rearrangements of the transmembrane helices necessary for optimal nucleotide hydrolysis.
To investigate the structural/functional roles of the conserved polar residues in the transmembrane helices of human NTPDase3, each was singly mutated to alanine. All mutants were properly glycosylated and had specific activities similar to wild-type, except Q44A. The Q44A mutation decreased specific activities by approximately 50%–70%, and nearly eliminated Triton X-100 detergent inhibition. The same conserved polar residues were mutated to cysteine, singly and in pairs, to allow a disulfide cross-linking strategy to map potential inter- and intra-molecular hydrogen bond interactions. The results support the centrality of Q44 for the strong inter-molecular interactions driving the association of the N-terminal domains of two NTPDase3 monomers in a dimer, while S39 and T495 may contribute to helical interactions involved in forming higher order oligomers. These results suggest a model for putative hydrogen bond interactions of the conserved polar residues in the transmembrane domain of native, dimeric NTPDase3 that are important for protein expression, activity, and susceptibility to membrane perturbations.
This dissertation provides additional insights to the structural elements that regulate the enzymatic activity of the NTPDases. With the recent elucidation of the crystal structure of the extracellular portion of rat NTPDase2, the major remaining structural questions regarding the cell membrane NTPDases are how the TM helices interact and how these interactions are coupled to modulation of enzyme activity and NTPDase function. This dissertation addresses these questions for human NTPDase3, and suggests that similar structure-function relationships are relevant to the family of NTPDases controlling purinergic signaling.