SUR2A is a member of the ABC transporter superfamily. SUR2A mediated regulation of KATP channels is essential as mutations in the nucleotide binding domains (NBDs) of SUR2A are associated with cardiovascular disorders. Studies of eukaryotic NBDs, such as SUR2A, are hindered by low solubility of the isolated domain. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the definition of the domain boundaries. Boundaries were initially predicted using a combination of a structure-based sequence alignment and homology modeling, and subsequently verified by testing the solubility of five SUR2A NBD1 constructs with different N- or C-terminal boundaries. The boundaries of SUR2A NBD1 essential for solubility were identified. CD and NMR data indicate that SUR2A NBD1 is folded. Our method may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins such as SUR isoforms, SUR2B and SUR2C, and the vacuolar transporter, Ycf1p.

ATP-sensitive potassium (KATP) channels have crucial roles in several biological processes. KATP channels possess four regulatory sulfonylurea receptors. The SUR proteins are members of the ubiquitous ATP-binding cassette (ABC) superfamily. However, unlike most ABC proteins, SURs do not transport substrates but function strictly as regulators of KATP channel activity. Currently, studies into the molecular basis by which various mutations in SUR2A cause disease are highly limited. This is primarily a consequence of poor solubility of isolated SUR2A NBDs, as is typical for many eukaryotic NBDs. By employing structure-based sequence alignments and biophysical studies, we determined domain boundaries for SUR2A NBD1 that enabled, for the first time, NMR studies of NBD1. Our biophysical studies demonstrate that the isolated SUR2A NBD1 is folded and exhibits differential dynamics upon ATP binding activity. Additional studies are now possible to examine the effects of disease-causing mutations on structure, dynamics, and interactions of NBD1.

SUR2A is a member of the ABC transporter superfamily. SUR2A mediated regulation of KATP channels is essential as mutations in the nucleotide binding domains (NBDs) of SUR2A are associated with cardiovascular disorders. Studies of eukaryotic NBDs, such as SUR2A, are hindered by low solubility of the isolated domain. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the definition of the domain boundaries. Boundaries were initially predicted using a combination of a structure-based sequence alignment and homology modeling, and subsequently verified by testing the solubility of five SUR2A NBD1 constructs with different N- or C-terminal boundaries. The boundaries of SUR2A NBD1 essential for solubility were identified. CD and NMR data indicate that SUR2A NBD1 is folded. Our method may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins such as SUR isoforms, SUR2B and SUR2C, and the vacuolar transporter, Ycf1p.

ATP-sensitive potassium (KATP) channels have crucial roles in several biological processes. KATP channels possess four regulatory sulfonylurea receptors. The SUR proteins are members of the ubiquitous ATP-binding cassette (ABC) superfamily. However, unlike most ABC proteins, SURs do not transport substrates but function strictly as regulators of KATP channel activity. Currently, studies into the molecular basis by which various mutations in SUR2A cause disease are highly limited. This is primarily a consequence of poor solubility of isolated SUR2A NBDs, as is typical for many eukaryotic NBDs. By employing structure-based sequence alignments and biophysical studies, we determined domain boundaries for SUR2A NBD1 that enabled, for the first time, NMR studies of NBD1. Our biophysical studies demonstrate that the isolated SUR2A NBD1 is folded and exhibits differential dynamics upon ATP binding activity. Additional studies are now possible to examine the effects of disease-causing mutations on structure, dynamics, and interactions of NBD1.

Cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC form the bacteriocin colicin V (ColV) secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter with nucleotide-binding motifs in the C-terminal domain (CTD). To study the role of CvaB-CTD in the ColV secretion, a truncated construct of this domain was made and over-expressed. Different forms of CvaB-CTD were obtained during purification, and were identified as monomer, dimer, and oligomer on gel filtration. Nucleotide binding was shown critical for the CvaB-CTD dimerization: oligomers could be converted into dimers by nucleotide bindings; the removal of nucleotide from dimers resulted in transient monomers followed by CTD oligomerization and aggregation; no dimer form could be cross-linked from the nucleotide-binding deficient mutant D654H. The spatial proximity of the Walker A site and ABC signature motif in CTD dimer was identified through disulfide cross-linking of mixed CvaB-CTD with mutants A530C and L630C, while mutations did not dimerize individually. Those results indicated that the CvaB-CTD formed a nucleotide-dependent head-to-tail dimer. Molecular basis of differential nucleotide bindings was also studied through bioinformatics prediction and biochemical verification. Through sequence alignment and homology modeling with bound ATP or GTP, it was found that the Ser503 and Gln504 on aromatic stacking region (Y501DSQ-loop) of CvaB-CTD provided two additional hydrogen-bonds to GTP, but not to ATP. Site-directed mutations of the S503A and/or Q504L were designed based on the model. While site-directed mutagenesis studies of Walker A&B sites or the ABC signature motif affected little on the GTP-binding preference, the double mutation (S503A/Q504L) on the Y501DSQ-loop increased both ATP-binding and ATPase activity at low temperatures. The double mutant showed slight decrease of GTP-binding and about 10-fold increase of the ATP/GTP-binding ratio. Similar temperature sensitivity in nucleotide-binding and activity assays were identified in the double mutant at the same time. Mutations on the Y501DSQ-loop did not affect the ColV secretion level in vivo. Together, the Y501DSQ-loop is structurally involved in the differential binding of GTP over ATP.