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1. Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence

   https://doi.org/10.1371/journal.pgen.1010323  

   Mohammadi, Shabnam ; Herrera-Álvarez, Santiago ; Yang, Lu ; Rodríguez-Ordoñez, María del ; Zhang, Karen ; Storz, Jay F. ; Dobler, Susanne ; Crawford, Andrew J. ; Andolfatto, Peter ( August 2022 , PLOS Genetics)

   Malik, Harmit S. (Ed.)

   A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the α-subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitution’s effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases (See S1 Text for Spanish translation). 

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2. FXYD proteins and sodium pump regulatory mechanisms

   https://doi.org/10.1085/jgp.202012633  

   Yap, John Q. ; Seflova, Jaroslava ; Sweazey, Ryan ; Artigas, Pablo ; Robia, Seth L. ( March 2021 , Journal of General Physiology)

   The sodium/potassium-ATPase (NKA) is the enzyme that establishes gradients of sodium and potassium across the plasma membrane. NKA activity is tightly regulated for different physiological contexts through interactions with single-span transmembrane peptides, the FXYD proteins. This diverse family of regulators has in common a domain containing a Phe-X-Tyr-Asp (FXYD) motif, two conserved glycines, and one serine residue. In humans, there are seven tissue-specific FXYD proteins that differentially modulate NKA kinetics as appropriate for each system, providing dynamic responsiveness to changing physiological conditions. Our understanding of how FXYD proteins contribute to homeostasis has benefitted from recent advances described in this review: biochemical and biophysical studies have provided insight into regulatory mechanisms, genetic models have uncovered remarkable complexity of FXYD function in integrated physiological systems, new posttranslational modifications have been identified, high-resolution structural studies have revealed new details of the regulatory interaction with NKA, and new clinical correlations have been uncovered. In this review, we address the structural determinants of diverse FXYD functions and the special roles of FXYDs in various physiological systems. We also discuss the possible roles of FXYDs in protein trafficking and regulation of non-NKA targets.

    

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3. Lipidomic Analysis of Arabidopsis T-DNA Insertion Lines Leads to Identification and Characterization of C-Terminal Alterations in FATTY ACID DESATURASE 6

   https://doi.org/10.1093/pcp/pcac088  

   Lusk, Hannah J. ; Neumann, Nicholas ; Colter, Madeline ; Roth, Mary R. ; Tamura, Pamela ; Yao, Libin ; Shiva, Sunitha ; Shah, Jyoti ; Schrick, Kathrin ; Durrett, Timothy P. ; et al ( June 2022 , Plant and Cell Physiology)

   Mass-spectrometry-based screening of lipid extracts of wounded and unwounded leaves from a collection of 364 Arabidopsis thaliana T-DNA insertion lines produced lipid profiles that were scored on the number and significance of their differences from the leaf lipid profiles of wild-type plants. The analysis identified Salk_109175C, which displayed alterations in leaf chloroplast glycerolipid composition, including a decreased ratio between two monogalactosyldiacylglycerol (MGDG) molecular species, MGDG(18:3/16:3) and MGDG(18:3/18:3). Salk_109175C has a confirmed insertion in the At5g64790 locus; the insertion did not co-segregate with the recessive lipid phenotype in the F2 generation of a wild-type (Columbia-0) × Salk_109175C cross. The altered lipid compositional phenotype mapped to the At4g30950 locus, which encodes the plastidial ω-6 desaturase FATTY ACID DESATURASE 6 (FAD6). Sequencing revealed a splice-site mutation, leading to the in-frame deletion of 13 amino acids near the C-terminal end of the 448 amino acid protein. Heterologous expression in yeast showed that this deletion eliminates desaturase activity and reduces protein stability. Sequence comparison across species revealed that several amino acids within the deletion are conserved in plants and cyanobacteria. Individual point mutations in four conserved residues resulted in 77–97% reductions in desaturase activity, while a construct with all four alanine substitutions lacked activity. The data suggest that the deleted region of FAD6, which is on the C-terminal side of the four putative transmembrane segments and the histidine boxes putatively involved in catalysis, is critical for FAD6 function.

    

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4. Evolution of the SARS‐CoV ‐2 proteome in three dimensions (3D) during the first 6 months of the COVID ‐19 pandemic

   https://doi.org/10.1002/prot.26250  

   Lubin, Joseph H. ; Zardecki, Christine ; Dolan, Elliott M. ; Lu, Changpeng ; Shen, Zhuofan ; Dutta, Shuchismita ; Westbrook, John D. ; Hudson, Brian P. ; Goodsell, David S. ; Williams, Jonathan K. ; et al ( October 2021 , Proteins: Structure, Function, and Bioinformatics)

   Understanding the molecular evolution of the SARS‐CoV‐2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three‐dimensional structures of SARS‐CoV‐2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID‐19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes. Catalytic residues in active sites and binding residues in protein–protein interfaces showed modest, but significant, numbers of substitutions, highlighting the mutational robustness of the viral proteome. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi‐Gaussian distribution. Detailed results are presented for potential drug discovery targets and the four structural proteins that comprise the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and protein–protein and protein–nucleic acid interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure‐based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

    

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5. Elucidating the Structural Impacts of Protein InDels

   https://doi.org/10.3390/biom12101435  

   Jilani, Muneeba ; Turcan, Alistair ; Haspel, Nurit ; Jagodzinski, Filip ( October 2022 , Biomolecules)

   The effects of amino acid insertions and deletions (InDels) remain a rather under-explored area of structural biology. These variations oftentimes are the cause of numerous disease phenotypes. In spite of this, research to study InDels and their structural significance remains limited, primarily due to a lack of experimental information and computational methods. In this work, we fill this gap by modeling InDels computationally; we investigate the rigidity differences between the wildtype and a mutant variant with one or more InDels. Further, we compare how structural effects due to InDels differ from the effects of amino acid substitutions, which are another type of amino acid mutation. We finish by performing a correlation analysis between our rigidity-based metrics and wet lab data for their ability to infer the effects of InDels on protein fitness. 

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