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A plethora of membrane proteins are found along the cell surface and on the convoluted labyrinth of membranes surrounding organelles. Since the advent of various structural biology techniques, a sub-population of these proteins has become accessible to investigation at near-atomic resolutions. The predominant bona fide methods for structure solution, X-ray crystallography and cryo-EM, provide high resolution in three-dimensional space at the cost of neglecting protein motions through time. Though structures provide various rigid snapshots, only an amorphous mechanistic understanding can be inferred from interpolations between these different static states. In this review, we discuss various techniques that have been utilized in observing dynamic conformational intermediaries that remain elusive from rigid structures. More specifically we discuss the application of structural techniques such as NMR, cryo-EM and X-ray crystallography in studying protein dynamics along with complementation by conformational trapping by specific binders such as antibodies. We finally showcase the strength of various biophysical techniques including FRET, EPR and computational approaches using a multitude of succinct examples from GPCRs, transporters and ion channels.

Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S. cerevisiae HOL1 mRNA is under translational control by polyamines, and we reveal that the encoded membrane transporter Hol1 is a high-affinity polyamine transporter and is required for yeast growth under limiting polyamine conditions. Moreover, we show that polyamine inhibition of the translation factor eIF5A impairs translation termination at a Pro-Ser-stop motif in a conserved upstream open reading frame on the HOL1 mRNA to repress Hol1 synthesis under conditions of elevated polyamines. Our findings reveal that polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels., Competing Interests: Declaration of interests T.E.D. is a member of the scientific advisory board at the journal Molecular Cell., (Published by Elsevier Inc.)

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

ATG9, the only transmembrane protein in the core macroautophagy/autophagy machinery, is a key player in the early stages of autophagosome formation. Yet, the lack of a high-resolution structure of ATG9 was a major impediment in understanding its three-dimensional organization and function. We recently solved a high-resolution cryoEM structure of the ubiquitously expressed human ATG9A isoform. The structure revealed that ATG9A is a domain-swapped homotrimer with a unique fold, and has an internal network of branched cavities. In cellulo analyses demonstrated the functional importance of the cavity-lining residues. These cavities could serve as conduits for transport of hydrophilic moieties, such as lipid headgroups, across the bilayer. Finally, structure-guided molecular dynamics predicted that ATG9A has membrane-bending properties, which is consistent with its localization to highly curved membranes.

Autophagy is a catabolic process involving capture of cytoplasmic materials into double-membraned autophagosomes that subsequently fuse with lysosomes for degradation of the materials by lysosomal hydrolases. One of the least understood components of the autophagy machinery is the transmembrane protein ATG9. Here, we report a cryoelectron microscopy structure of the human ATG9A isoform at 2.9-Å resolution. The structure reveals a fold with a homotrimeric domain-swapped architecture, multiple membrane spans, and a network of branched cavities, consistent with ATG9A being a membrane transporter. Mutational analyses support a role for the cavities in the function of ATG9A. In addition, structure-guided molecular simulations predict that ATG9A causes membrane bending, explaining the localization of this protein to small vesicles and highly curved edges of growing autophagosomes., Competing Interests: Declaration of Interests The authors declare no competing interest., (Published by Elsevier Inc.)

Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella -containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe 2+ , with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe 2+ , Mn 2+ , Co 2+ , or Zn 2+ Consequently, flooding infected cells with either Mn 2+ or Zn 2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila Δ mavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection., Competing Interests: The authors declare no conflict of interest.

Iron is universally important to cellular metabolism, and mitoferrin-1 and -2 have been proposed to be the iron importers of mitochondria, the cell's assembly plant of heme and iron-sulfur clusters. These iron-containing prosthetic groups are critical for a host of physiological processes ranging from oxygen transport and energy consumption to maintaining protein structural integrity. Mitoferrin-1 (Mfrn1) belongs to the mitochondrial carrier (MC) family and is atypical given its putative metallic cargo; most MCs transport nucleotides, amino acids, or other small- to medium-size metabolites. Despite the clear importance of Mfrn1 in iron utilization, its transport activity has not been demonstrated unambiguously. To bridge this knowledge gap, we have purified recombinant Mfrn1 under non-denaturing conditions and probed its metal ion-binding and transport functions. Isothermal titration calorimetry indicates that Mfrn1 has micromolar affinity for Fe(II), Mn(II), Co(II), and Ni(II). Mfrn1 was incorporated into defined liposomes, and iron transport was reconstituted in vitro , demonstrating that Mfrn1 can transport iron. Mfrn1 can also transport manganese, cobalt, copper, and zinc but discriminates against nickel. Experiments with candidate ligands for cellular labile iron reveal that Mfrn1 transports free iron and not a chelated iron complex and selects against alkali divalent ions. Extensive mutagenesis identified multiple residues that are crucial for metal binding, transport activity, or both. There is a clear abundance of residues with side chains that can coordinate first-row transition metal ions, suggesting that these could form primary or auxiliary metal-binding sites during the transport process.

Cytolytic peptides have commanded attention for their anticancer potential because the membrane-disrupting function that produces cell death is less likely to be overcome by resistant mutations. Congruently, peptides that are involved in molecular recognition and biological activities become attractive therapeutic candidates because of their high specificity, better affinity, reduced immunogenicity, and reduced off target toxicity. However, problems of inadequate delivery, rapid deactivation in vivo, and poor bioavailability have limited clinical application. Therefore, peptide drug development for clinical use requires an appropriate combination of an effective therapeutic peptide and a robust delivery methodology. In this chapter, we describe methods for the postformulation insertion of peptide drugs into lipidic nanostructures, the physical characterization of peptide-nanostructure complexes, and the evaluation of their therapeutic effectiveness both in vitro and in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation with detergents, such as n-octyl glucoside. During BAX activation with n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.