*Article* **Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model**

**Flora Szeri 1,2 , Valentina Corradi 3 , Fatemeh Niaziorimi 1 , Sylvia Donnelly 1 , Gwenaëlle Conseil 4 , Susan P. C. Cole 4 , D. Peter Tieleman <sup>3</sup> and Koen van de Wetering 1, \***


**Abstract:** Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC<sup>4</sup> ), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC<sup>4</sup> binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC<sup>4</sup> binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC<sup>4</sup> -binding site.

**Keywords:** ABC transporter; pseudoxanthoma elasticum; homology modeling; substrate-binding site; cellular ATP efflux; mutagenesis
