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1. Roles of conserved tryptophans in trimerization of HIV‐1 membrane‐proximal external regions: Implications for virucidal design via alchemical free‐energy molecular simulations

   https://doi.org/10.1002/prot.25504  

   Gossert, Steven T. ; Parajuli, Bibek ; Chaiken, Irwin ; Abrams, Cameron F. ( April 2018 , Proteins: Structure, Function, and Bioinformatics)

   The Dual‐Action Virolytic Entry Inhibitors, or “DAVEI's,” are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15‐residue fragment from the membrane‐proximal external region (MPER) of HIV‐1 gp41. DAVEI's trigger rupture of HIV‐1 virions, and the interaction site between DAVEI MPER and HIV‐1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free‐energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER₃. We found that a W666A mutation had a large positivefor all three protomers, while W672A had a large positivefor only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER₃stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER₃. Our findings are consistent with our previous experimental study of W‐to‐A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV‐1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.

    

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2. GreenCut protein CPLD 49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b 6 f complex accumulation

   https://doi.org/10.1111/tpj.13915  

   Wittkopp, Tyler M. ; Saroussi, Shai ; Yang, Wenqiang ; Johnson, Xenie ; Kim, Rick G. ; Heinnickel, Mark L. ; Russell, James J. ; Phuthong, Witchukorn ; Dent, Rachel M. ; Broeckling, Corey D. ; et al ( May 2018 , The Plant Journal)

   The GreenCut encompasses a suite of nucleus‐encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non‐photosynthetic/heterotrophic organisms. InChlamydomonas reinhardtii,CPLD49 (Conserved inPlantLineage andDiatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that acpld49mutant has impaired photoautotrophic growth under high‐light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochromeb₆fcomplex (Cytb₆f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore,CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein,CPLD38; a mutant null forCPLD38 also impacts Cytb₆fcomplex accumulation. We investigated several potential functions ofCPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis thatCPLD38 andCPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb₆fcomplex. Based on motifs ofCPLD49 and the activities of otherCPLD49‐like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb₆f.

    

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3. Structural considerations of folded protein import through the chloroplast TOC / TIC translocons

   https://doi.org/10.1002/1873-3468.13342  

   Ganesan, Iniyan ; Theg, Steven M. ( March 2019 , FEBS Letters)

   Protein import into chloroplasts is carried out by the protein translocons at the outer and inner envelope membranes (TOCandTIC). Detailed structures for these translocons are lacking, with only a low‐resolutionTOCcomplex structure available. Recently, we showed that theTOC/TICtranslocons can import folded proteins, a rather unique feat for a coupled double membrane system. We also determined the maximum functionalTOC/TICpore size to be 30–35 Å. Here, we discuss how such large pores could form and compare the structural dynamics of the pore‐forming Toc75 subunit to its bacterial/mitochondrial Omp85 family homologs. We put forward structural models that can be empirically tested and also briefly review the pore dynamics of other protein translocons with known structures.

    

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4. Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH , Ca 2+ and GABA

   https://doi.org/10.1111/nph.15863  

   Domingos, Patrícia ; Dias, Pedro N. ; Tavares, Bárbara ; Portes, Maria Teresa ; Wudick, Michael M. ; Konrad, Kai R. ; Gilliham, Matthew ; Bicho, Ana ; Feijó, José A. ( May 2019 , New Phytologist)

   We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth inArabidopsis thaliana(Col‐0).

   Patch‐clamp whole‐cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca²⁺]_cyt). We investigated the pollen‐expressed proteinsAtSLAH3,AtALMT12,AtTMEM16 andAtCCCas the putative anion transporters responsible for these currents.

   AtCCC‐GFPwas observed at the shank andAtSLAH3‐GFPat the tip and shank of thePTplasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip ofPTs with an anion vibrating probe were significantly lower inslah3^−/−andccc^−/−mutants, but unaffected inalmt12^−/−andtmem16^−/−. We further characterised the effect ofpHandGABAby patch clamp. Strong regulation by extracellularpHwas observed in the wild‐type, but not intmem16^−/−. Our results are compatible withAtTMEM16 functioning as an anion/H⁺cotransporter and therefore, as a putativepHsensor.GABApresence: (1) inhibited the overall currents, an effect that is abrogated in thealmt12^−/−and (2) reduced the current inAtALMT12 transfectedCOS‐7 cells, strongly suggesting the direct interaction ofGABAwithAtALMT12.

   Our data show thatAtSLAH3 andAtCCCactivity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linkingPTgrowth modulation bypH,GABA, and [Ca²⁺]_cytthrough anionic transporters.

    

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5. The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

   https://doi.org/10.1371/journal.pbio.3001569  

   Yoon, Jimin ; Nekongo, Emmanuel E. ; Patrick, Jessica E. ; Hui, Tiffani ; Phillips, Angela M. ; Ponomarenko, Anna I. ; Hendel, Samuel J. ; Sebastian, Rebecca M. ; Zhang, Yu Meng ; Butty, Vincent L. ; et al ( February 2022 , PLOS Biology)

   Malik, Harmit S. (Ed.)

   The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation. 

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