Functional hot spots in human ATP-binding cassette transporter nucleotide binding domains

Abstract

The human ATP-binding cassette (ABC) transporter superfamily consists of 48 integral membrane proteins that couple the action of ATP binding and hydrolysis to the transport of diverse substrates across cellular membranes. Defects in 18 transporters have been implicated in human disease. In hundreds of cases, disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs). The functional impact of the majority of ABC transporter nsSNPs has yet to be experimentally characterized. Here, we combine experimental mutational studies with sequence and structural analysis to describe the impact of nsSNPs in human ABC transporters. First, the disease associations of 39 nsSNPs in 10 transporters were rationalized by identifying two conserved loops and a small α-helical region that may be involved in interdomain communication necessary for transport of substrates. Second, an approach to discriminate between disease-associated and neutral nsSNPs was developed and tailored to this superfamily. Finally, the functional impact of 40 unannotated nsSNPs in seven ABC transporters identified in 247 ethnically diverse individuals studied by the Pharmacogenetics of Membrane Transporters consortium was predicted. Three predictions were experimentally tested using human embryonic kidney epithelial (HEK) 293 cells stably transfected with the reference multidrug resistance transporter 4 and its variants to examine functional differences in transport of the antiviral drug, tenofovir. The experimental results confirmed two predictions. Our analysis provides a structural and evolutionary framework for rationalizing and predicting the functional effects of nsSNPs in this clinically important membrane transporter superfamily.

Figure 1

Functional hot spots and experimentally characterized nsSNPs mapped to the nucleotide binding domain (NBD) sequences of human MRP4. The previously identified nucleotide-binding loop and the ABC signature motif are indicated in black. C-loop 1, the ARA motif, and C-loop 2 are indicated in blue. Experimentally characterized nsSNPs, G487E and K498E (NBD1), and V1071I (NBD2) are indicated in red.

Figure 2

Disease-associated nsSNPs at a putative communication network between the NBD and the transmembrane helices of human ABC transporters. Mutations in the ARA motif, which forms a small, partially buried α-helix that may interact with the transmembrane helices, are associated with disease in seven human ABC transporters (Table I). A surface representation of the human ABCC NBD2 model structure is shown with the ARA motif colored in red, the mobile Q-loop in purple, and the TMD of the structure of the Staphylococcus aureus multidrug ABC transporter Sav1866 in light blue. The transmembrane segment that appears to interact with the Q-loop/ARA region is a portion of the L2 loop.²

Figure 3

An intracellular surface region with conserved disease associations. C-loop 2 is shown in purple, mapped on the comparative models of NBD1 (dark grey) and NBD2 (light grey) in human P-gp (ABCB1). The previously identified functional histidine is shown in yellow. An alignment of sequences containing disease-associated mutations is shown at right with residue coloring indicating sequence conservation; each disease-associated nsSNP is indicated with a red star (Table II).

Figure 4

Performance of random forest classifier on a test set of 72 cystic fibrosis mutations. (A) Features used for discriminating between disease-associated and neutral nsSNPs. (B) A receiver-operator curve (ROC) showing the true-positive and false-positive rates for the clinical, experimental, and ABC transporter-trained random forests on the cystic fibrosis test set at left. The clinically trained random forest performs best on both the cystic fibrosis test set and the ABC transporter test set.

Figure 5

Structural models of MRP4 nsSNPs G487E, K498E, and V1071I. The models for G487E and K498E were based on the crystallographic structure of the human multidrug resistance protein MRP1 (PDB ID: 2CBZ). The model for V1071I was based on the structure of a multidrug resistance protein from Plasmodium yoelii (PDB ID: 2GHI). ATP coordinates are based on a structural alignment of the MRP4 models with the dimeric structure of two ABC NBDs from Methanocaldococcus jannaschii (PDB ID: 1L2T). Residues within 3.5 Å of the cognate NBD are colored light blue. Residues within 3.5 Å of an ATP molecule are colored yellow. An interactive view is available in the electronic version of the article. PRO491 Figure 5

Figure 6

Efflux of [³H]-tenofovir by HEK cells expressing the wild type MRP4 and its variants. HEK293 cells stably expressing empty pcDNA5/FRT vector, the MRP4 wild type (reference), or its variants were preincubated with 1 μM [³H]-tenofovir disoproxil for 2 h under ATP-depleting conditions. After washing, the cells were supplemented with complete DMEM, and extracellular concentrations of tenofovir were determined at 0, 30, and 90 min. Data are mean ± standard deviation of triplicate determinations from one experiment and are representative of multiple experiments.

Figure 7

Effect of MRP4 variants on transport of [³H]-tenofovir. The percentage of tenofovir efflux (90 min) for each variant relative to the MRP4 reference was determined. Data are mean ± standard deviation calculated from nine to 22 determinations. Significant differences between the reference and the variants were assessed by one-way ANOVA and Bonferroni's multiple comparison test (*P < 0.05, **P < 0.001).