Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters.

ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.

Ligand specificity and affinity in the sulforhodamine B binding RNA aptamer.

Binding affinity and selectivity are critical properties of aptamers that must be optimized for any application. The sulforhodamine B binding RNA aptamer (SRB-2) is a somewhat promiscuous aptamer that can bind ligands that vary markedly in shape, size and charge. Here we categorize potential ligands based on their binding mode and structural characteristics required for high affinity and selectivity. Several known and potential ligands of SRB-2 were screened for binding affinity using LSPR, ITC and NMR spectroscopy. The study shows that rhodamine B has the ideal structural and electrostatic properties for selective and high-affinity binding of the SRB-2 aptamer.

Intrinsically disordered regions regulate the activities of ATP binding cassette transporters.

ATP binding cassette (ABC) proteins are a large family of membrane proteins present in all kingdoms of life. These multi-domain proteins are comprised, at minimum, of two membrane-spanning domains (MSD1, MSD2) and two cytosolic nucleotide binding domains (NBD1, NBD2). ATP binding and hydrolysis at the NBDs enables ABC proteins to actively transport solutes across membranes, regulate activities of other proteins, or function as channels. Like most eukaryotic membrane proteins, ABC proteins contain intrinsically disordered regions (IDRs). These conformationally dynamic regions in ABC proteins possess residual structure, are sites of phosphorylation, and mediate protein-protein interactions. Here, we review the role of IDRs in regulating ABC protein activity.

HK97 gp74 Possesses an α-Helical Insertion in the ßßα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities.

The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ßßα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two ß-strands of the ßßα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ßßα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle.IMPORTANCE This study demonstrates that residues outside the canonical ßßα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.