ABC transporters are polytopic proteins. ATP hydrolysis and substrate transport take place in separate domains, and these activities must be coordinated through a signal interface. We previously characterized a mutation (S558Y) in the yeast multidrug transporter Pdr5 that uncouples ATP hydrolysis and drug transport. To characterize the transmission interface, we used a genetic screen to isolate second-site mutations of S558Y that restore drug transport. We recovered suppressors that restore drug resistance; their locations provide functional evidence for an interface in the cis rather than the trans configuration indicated by structural and crosslinking studies of bacterial and eukaryotic efflux transporters. One mutation, E244G, defines the Q-loop of the deviant portion of NBD1, which is the hallmark of this group of fungal transporters. When moved to an otherwise wild-type background, this mutation and its counterpart in the canonical ATP-binding site Q951G show a similar reduction in drug resistance and in the very high basal-level ATP hydrolysis characteristic of Pdr5. A double E244G, Q951G mutant is considerably more drug sensitive than either of the single mutations. Surprisingly, then, the deviant and canonical Q-loop residues are functionally overlapping and equivalent in a strikingly asymmetric ABC transporter.

bPdr5 is a major ATP-binding cassette (ABC) multidrug transporter regarded as the founding member of a fungal subfamily of clinically significant efflux pumps. When these proteins are overexpressed, they confer broad-spectrum ultraresistance. To better understand the evolution of these proteins under selective pressure, we exposed a Saccharomyces cerevisiae yeast strain already overexpressing Pdr5 to a lethal concentration of cycloheximide. This approach gave mutations that confer greater resistance to a subset of transport substrates. One of these mutations, V656L, is located in intracellular loop 2 (ICL2), a region predicted by structural studies with several other ABC transporters to play a critical role in the transmission interface between the ATP hydrolysis and drug transport domains. We show that this mutation increases drug resistance, possibly by altering the efficiency with which the energy from ATP hydrolysis is used for transport. Val-656 is a conserved residue, and an alanine substitution creates a nearly null phenotype for drug transport as well as reduced ATPase activity. We posit that despite its unusually small size, ICL2 is part of the transmission interface, and that alterations in this pathway can increase or decrease resistance to a broad spectrum of drugs. The World Health Organization lists antimicrobial resistance as a major global concern that threatens advancements in medicine, national security, and economic development (http: //www.who.int/drugresistance/en/). Multidrug resistance remains a major clinical problem in the treatment of cancer and pathogenic infection (1). This phenomenon is attributable, in part, to the overexpression of ATP-binding cassette (ABC) efflux pumps that were initially identified and characterized in mammalian cells (2-5). Multidrug resistance was observed in Saccharomyces cerevisiae yeast in 1973, when it was attributed to a single-gene alteration (6), but the cloning and characterization of the efflux pumps mediating this phenomenon occurred much later (7-12).Pdr5 is a highly promiscuous ABC transporter. Overexpression leads to hyperresistance for all known substrates (13-15). This efflux pump has a basic structure found in full-length members of this superfamily. It has two nucleotide-binding domains and two transmembrane domains (TMDs) made up of six alpha helices, each of which is connected by intra-and extracellular loops. In several significant ways, however, Pdr5 departs from the canonical architecture of ABC transporters. It has a deviant ATPbinding site that lacks an obvious catalytic residue in the Walker B motif; it also contains deviant sequences in the Walker A, signature region (C-loop) and Q-loop motifs. Furthermore, in contrast to most ABC transporters, the intracellular loops (ICLs) are very short. ICL2 and ICL4 are predicted to have only 10 and 7 residues, respectively (16). Important studies with Tap1/Tap2 antigen transporter (17) and P-gp (18) using cross-linking of cysteinemodified residues provide evidence that the X-loop, Q-loop, and in...

A "universal" platform that can rapidly generate multiplex vaccine candidates is critically needed to control pandemics. Using the severe acute respiratory syndrome coronavirus 2 as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates was engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers was found to be the most potent vaccine candidate in animal models. Without any adjuvant, this vaccine stimulated robust immune responses, both T helper cell 1 (T H 1) and T H 2 immunoglobulin G subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new nanovaccine design framework might allow the rapid deployment of effective adjuvant-free phage-based vaccines against any emerging pathogen in the future.