The ABC exporter IrtAB imports and reduces mycobacterial siderophores.

Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin2,3. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain5. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.

Studying membrane proteins with MicroED.

The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.

Lipid flipping in the omega-3 fatty-acid transporter.

Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions to microcephaly. Mfsd2a transports long-chain unsaturated fatty-acids, including DHA and α-linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with the recently determined structures of Mfsd2a, the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remains unclear. Here, we report five single-particle cryo-EM structures of Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and displaying lipid-like densities modeled as ALA-LPC at four distinct positions. These Mfsd2a snapshots detail the flipping mechanism for lipid-LPC from outer to inner membrane leaflet and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with disease.

A recombinant platform to characterize the role of transmembrane protein hTMEM205 in Pt(II)-drug resistance and extrusion.

Platinum-coordination complexes are among the most effective chemotherapeutic drugs used in clinics for the treatment of cancer. Despite their efficacy, cancer cells can develop drug resistance leading to treatment failure and relapse. Cellular uptake and extrusion of Pt(ii)-complexes mediated by transmembrane proteins are critical in controlling the intracellular concentration of Pt(ii)-drugs and in developing pre-target resistance. TMEM205 is a human transmembrane protein (hTMEM205) overexpressed in cancer cells that are resistant to cisplatin, but its molecular function underlying - resistance remains elusive. We developed a low-cost and high-throughput recombinant expression platform coupled to in vivo functional resistance assays to study the molecular mechanism by which the orphan hTMEM205 protects against Pt(ii)-complex toxicity. Based on the original observation by the Rosenberg group, which led to the discovery of cisplatin, we performed quantitative analysis of the effects of Pt(ii)-coordination complexes on cellular growth and filamentation in E. coli cells expressing hTMEM205. By coupling our methods with Pt quantification and cellular profiling in control and hTMEM205-expressing cells, we demonstrate that hTMEM205 mediates Pt(ii)-drug export selectively towards cisplatin and oxaliplatin but not carboplatin. By mutation analysis, we reveal that hTMEM205 recognizes and allows Pt(ii)-extrusion by a putative sulfur-based translocation mechanism, thereby resulting in pre-target resistance. Thus, hTMEM205 represents a new potential target that can be exploited to reduce cellular resistance towards Pt(ii)-drugs.

Coordination promiscuity guarantees metal substrate selection in transmembrane primary-active Zn2+ pumps.

Metal selectivity in P1B-type ATPase pumps appears to be determined by amino acid motifs on their transmembrane helices. We reveal the principles governing substrate promiscuity towards first-, second- and third-row transition metals in a transmembrane Zn2+/Cd2+/Hg2+/Pb2+ P-type ATPase (ZntA), by dissecting its coordination chemistry. Atomic resolution characterization in detergent micelles and lipid bilayers reveals a "plastic" transmembrane metal-binding site that selects substrates by unique and diverse, yet defined, coordination geometries and ligand-metal distances.