ATP-binding cassette (ABC) exporters actively move chemically diverse substrates across biological membranes. Their malfunction leads to human diseases. Many ABC exporters encompass asymmetric nucleotide-binding sites (NBSs), and some of them are inhibited by the transported substrate. The functional relevance of the catalytic asymmetry or the mechanism for trans-inhibition remains elusive. Here, we investigated TmrAB, a functional homologue of the human antigen translocation complex TAP using advanced electron-electron double resonance spectroscopy. In the presence of ATP, the heterodimeric ABC exporter exists in a tunable equilibrium between inward- and outward-facing conformations. The two NBSs exhibit pronounced asymmetry in the open-to-close equilibrium. The closed conformation is more favored at the degenerate NBS, and closure of either of the NBS is sufficient to open the extracellular gate. We define the mechanistic basis for trans-inhibition, which operates by a reverse transition from the outward-facing state through an occluded conformation. These novel findings uncover the central role of reversible conformational equilibrium in the function and regulation of an ABC exporter and establish a mechanistic framework for future investigations on other medically important transporters with imprinted asymmetry. Also, this study demonstrates for the first-time the feasibility to resolve equilibrium populations at multiple domains and their interdependence for global conformational changes in a large membrane protein complex.

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.

Using engineered nanobodies with bright organic dyes (fluorescent nanobodies) and subsequent microfluidic cell manipulation, controlled nanobody delivery was achieved, allowing the multiplexed imaging and super-resolution of endogenous protein networks in living cells.

By designing and engineering photo-conditional viral inhibitors, spatiotemporal control of the transporter associated with antigen processing TAP was sustained, allowing the on-demand antigen translocation in human immune cell lines and primary cells by light.

1The visualization of endogenous proteins in living cells is a major challenge. A fundamental 2 requirement for spatiotemporally precise imaging is a minimal disturbance of protein function at 3 high signal-to-background ratio. Current approaches for visualization of native proteins in living 4 cells are limited by dark emitting, bulky fluorescent proteins and uncontrollable expression levels. 5Here, we demonstrate the labeling of endogenous proteins using nanobodies with site-specifically 6 engineered bright organic fluorophores, named fluorobodies. Their fast and fine-tuned intracellular 7 transfer by microfluidic cell squeezing allowed for low background, low toxicity, and high-8 throughput. Multiplexed imaging of distinct cellular structures was facilitated by specific protein 9targeting, culminating in live-cell super-resolution imaging of protein networks. The high-10 throughput delivery of engineered nanobodies will open new avenues in visualizing native cellular 11 structures with unprecedented accuracy in cell-based screens.