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1. Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase

   https://doi.org/10.1038/s41598-019-50009-2  

   Nielsen, Hang N. ; Spontarelli, Kerri ; Holm, Rikke ; Andersen, Jens Peter ; Einholm, Anja P. ; Artigas, Pablo ; Vilsen, Bente ( September 2019 , Scientific Reports)

   Three Na⁺sites are defined in the Na⁺-bound crystal structure of Na⁺, K⁺-ATPase. Sites I and II overlap with two K⁺sites in the K⁺-bound structure, whereas site III is unique and Na⁺specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na⁺at site III, but does not contribute to K⁺coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na⁺, K⁺-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na⁺and K⁺binding properties. Remarkably, Q925 substitutions had minor effects on Na⁺binding from the intracellular side of the membrane – in fact, mutations Q925A and Q925G increased the apparent Na⁺affinity – but caused dramatic reductions of the binding of K⁺as well as Na⁺from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na⁺-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na⁺at the extracellular side.

    

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2. A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities

   https://doi.org/10.1073/pnas.2313999120  

   Artigas, Pablo ; Meyer, Dylan J. ; Young, Victoria C. ; Spontarelli, Kerri ; Eastman, Jessica ; Strandquist, Evan ; Rui, Huan ; Roux, Benoît ; Birk, Matthew A. ; Nakanishi, Hanayo ; et al ( December 2023 , Proceedings of the National Academy of Sciences)

   Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na⁺,K⁺-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na⁺in exchange for 2 K⁺per hydrolyzed ATP.Artemiaexpress several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2_KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopusα1’s Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found thatArtemiaexpress two salinity-independent canonical α subunits (α1_NNand α3_NN), as well as two β variants, in addition to the salinity-controlled α2_KK. These β subunits permitted heterologous expression of the α2_KKpump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2_KKpumps and compared it to that ofXenopusα1 (and its α2_KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2_KK-like characteristics toXenopusα1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2_KK’s Lys308 helps to maintain high affinity for external K⁺when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K⁺-congener⁸⁶Rb⁺, we prove that double-lysine-substituted pumps transport 2Na⁺and 1 K⁺per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowingArtemiato maintain steeper Na⁺gradients in hypersaline environments.

    

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3. Na+,K+-ATPase with Disrupted Na+ Binding Sites I and III Binds Na+ with Increased Affinity at Site II and Undergoes Na+-Activated Phosphorylation with ATP

   https://doi.org/10.3390/biom14010135  

   Nielsen, Hang N. ; Holm, Rikke ; Sweazey, Ryan ; Andersen, Jens Peter ; Artigas, Pablo ; Vilsen, Bente ( January 2024 , Biomolecules)

   Na+,K+-ATPase actively extrudes three cytoplasmic Na+ ions in exchange for two extracellular K+ ions for each ATP hydrolyzed. The atomic structure with bound Na+ identifies three Na+ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na+ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na+ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na+ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na+ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na+ affinity. This indicates that Na+ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E1-like configuration, allowing Na+ but not K+ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.

    

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4. Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway

   https://doi.org/10.1128/JVI.01384-17  

   Blackburn, Brody J. ; Li, Shuaizhi ; Roznowski, Aaron P. ; Perez, Alexis R. ; Villarreal, Rodrigo H. ; Johnson, Curtis J. ; Hardy, Margaret ; Tuckerman, Edward C. ; Burch, April D. ; Fane, Bentley A. ; et al ( November 2017 , Journal of Virology)

   ABSTRACT Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability. IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway. 

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5. Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids

   https://doi.org/10.1021/acs.jpcb.3c03791  

   Friedman, Anika J. ; Padgette, Hannah M. ; Kramer, Levi ; Liechty, Evan T. ; Donovan, Gregory W. ; Fox, Jerome M. ; Shirts, Michael R. ( October 2023 , The Journal of Physical Chemistry B)

   Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L–11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a β site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the β site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments). 

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