Mutations in SCN4A gene encoding Nav1.4 channel α-subunit, are known to cause neuromuscular disorders such as myotonia or paralysis. Here, we study the effect of two amino acid replacements, K1302Q and G1306E, in the DIII-IV loop of the channel, corresponding to mutations found in patients with myotonia. We combine clinical, electrophysiological, and molecular modeling data to provide a holistic picture of the molecular mechanisms operating in mutant channels and eventually leading to pathology. We analyze the existing clinical data for patients with the K1302Q substitution, which was reported for adults with or without myotonia phenotypes, and report two new unrelated patients with the G1306E substitution, who presented with severe neonatal episodic laryngospasm and childhood-onset myotonia. We provide a functional analysis of the mutant channels by expressing Nav1.4 α-subunit in Xenopus oocytes in combination with β1 subunit and recording sodium currents using two-electrode voltage clamp. The K1302Q variant exhibits abnormal voltage dependence of steady-state fast inactivation, being the likely cause of pathology. K1302Q does not lead to decelerated fast inactivation, unlike several other myotonic mutations such as G1306E. For both mutants, we observe increased window currents corresponding to a larger population of channels available for activation. To elaborate the structural rationale for our experimental data, we explore the contacts involving K/Q1302 and E1306 in the AlphaFold2 model of wild-type Nav1.4 and Monte Carlo-minimized models of mutant channels. Our data provide the missing evidence to support the classification of K1302Q variant as likely pathogenic and may be used by clinicians.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that has been the main cause of diarrhea in piglets since 2010 in China. The aim of this study was to investigate sequence variation and recombination events in the spike (S) gene of PEDV isolates from China. Thirty complete S gene sequences were obtained from PEDV-positive samples collected in six provinces in China from 2020 to 2023. Phylogenetic analysis showed that 10% (3/30) belonged to subtype GII-a, 6.67% (2/30) were categorized as subtype GII-b, 66.67% (20/30) were categorized as subtype GII-c, and 16.66% (5/30) were clustered with the S-INDEL strains. Amino acid sequence alignments showed that, when compared to strains of other subtypes, the GII-c strains had two characteristic amino acid substitutions (N139D and I289M). Five S-INDEL subtype strains had a single amino acid deletion (139N) and four amino acid substitutions (N118G, T137S, A138S, and D141G). Recombination analysis allowed six putative recombination events to be identified, one involving recombination between GII-c strains, two involving GII-c and GII-b strains, two involving GII-c and GI-a strains, and one involving GII-a and GI-b strains. These results suggest that recombination between PEDV strains has been common and complex in recent years and is one of the main reasons for the continuous variation of PEDV strains.

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The x-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4% and 7% (ΔG ~ 1.5-1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer (ΔG ~ 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a β-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble β-glucans, such as cellulose, and helps fold CD. To determine how aromatic residues contribute to proper folding and enzyme activity, we performed alanine scanning for 32 aromatic residues in the BD. The study did not identify a single residue involved in glucan binding. However, several aromatic residues were found to be involved in BD or CD folding and in modulating the activity of the full-length enzyme. Among those aromatic residue mutations, the W43A mutation led to reduced solubility of the BD and full-length protein and resulted in a full-length enzyme with significantly lower activity. The activity of W43F and W43Y was significantly higher than that of W43A. In addition, Ala substitutions of Tyr83, Tyr113, and Tyr17 led to a reduction in trehalase activity, but Phe substitutions of these residues could be tolerated, as these mutants maintained activities similar to WT activity. Thus, these aromatic residues in BD may interact with CD and modulate enzyme activity. KEY POINTS: • Aromatic residues in the BD are involved in BD and CD folding. • Aromatic residues in the BD near the CD active site modulate enzyme activity. • BD interacts with CD and closely modulates enzyme activity.

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Human hyaluronidase 1 (HYAL1) and PH20 play vital roles in degrading hyaluronic acids through the substrate-assisted double displacement mechanism. While HYAL1, a lysosomal enzyme, functions optimally under acidic conditions, PH20, a sperm surface hyaluronidase, displays a broader pH range, from acidic to neutral. Our objective was to extend HYAL1\'s pH range towards neutral pH by introducing repulsive charge-charge interactions involving the catalytic Glu131, increasing its pKa as the proton donor. Substituting individual acidic residues in the β3-loop (S77D), β3\'-β3″ hairpin (T86D and P87E), and at Ala132 (A132D and A132E) enabled HYAL1 to demonstrate enzyme activity at pH 7, with the mutants S77D, P87E, and A132E showing the highest activity in the substrate gel assay. However, double and triple substitutions, including S77D/T86D/A132E as found in the PH20 configuration, did not result in enhanced activity compared to single substitutions. Conversely, PH20 mutants with non-acidic substitutions, such as D94S in the β3-loop and D103T in the β3\'-β3″ hairpin, significantly reduced activity within the pH range of 4 to 7. However, the PH20 mutant E149A, reciprocally substituted compared to A132E in HYAL1, exhibited activity similar to PH20 wild-type (WT) at pH 7. In a turbidimetric assay, HYAL1 mutants with single acidic substitutions exhibited activity similar to that of PH20 WT at pH 7. These results suggest that substituting acidic residues near Glu131 results in HYAL1 activity at neutral pH through electrostatic repulsion. This study highlights the significance of charge-charge interactions in both HYAL1 and PH20 in regulating the pH-dependent activity of hyaluronidases.

Most cases of human prion disease arise due to spontaneous misfolding of WT or mutant prion protein, yet recapitulating this event in animal models has proven challenging. It remains unclear whether spontaneous prion generation can occur within the mouse lifespan in the absence of protein overexpression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knockin mice that express the misfolding-prone bank vole prion protein (BVPrP). While mice expressing WT BVPrP (I109 variant) remained free from neurological disease, a subset of mice expressing BVPrP with mutations (D178N or E200K) causing genetic prion disease developed progressive neurological illness. Brains from spontaneously ill knockin mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant BVPrP. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP-knockin mice exhibited prion seeding activity and transmitted disease to mice expressing WT BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical before and after transmission, suggesting that both mutations guide the formation of a similar atypical prion strain. These findings imply that knockin mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action.

Pyrethroids are widely used against agricultural pests and human disease vectors due to their broad insecticidal spectrum, fast action, and low mammalian toxicity. Unfortunately, overuse of pyrethroids has led to knockdown resistance (kdr) caused by mutations in voltage-gated sodium channels. Mutation I1011M was repeatedly detected in numerous pyrethroid-resistant Aedes aegypti populations from Latin American and Brazil. In addition, mutation G923V was first reported to coexist with I1011M in permethrin/DDT-resistant Ae. aegypti, whether G923V enhances the I1011M-mediated pyrethroid resistance in sodium channels remains unclear. In this study, we introduced mutations G923V and I1011M alone or in combination into the pyrethroid-sensitive sodium channel AaNav1-1 and examined the effects of these mutations on gating properties and pyrethroid sensitivity. We found mutations I1011M and G923V + I1011M shifted the voltage dependence of activation in the depolarizing direction, and none of mutations affect the voltage-dependence of inactivation. G923V and G923V + I1011M mutations reduced the channel sensitivity to both Type I and Type II pyrethroids. However, I1011M alone conferred resistance to Type I pyrethroids, not to Type II pyrethroids. Interestingly, significant synergism effects on Type I pyrethroids were observed between mutations G923V and I1011M. The effects of all mutations on channel sensitivity to DDT were identical with those to Type I pyrethroids. Our results confirm the molecular basis of resistance mediated by mutations G923V and I1011M and may contribute to develop molecular markers for monitoring pest resistance to pyrethroids.

Rice blast, caused by Magnaporthe oryzae, is a devastating fungal disease worldwide. Pydiflumetofen (Pyd) is a new succinate dehydrogenase inhibitor (SDHI) that exhibited anti-fungal activity against M. oryzae. However, control of rice blast by Pyd and risk of resistance to Pyd are not well studied in this pathogen. The baseline sensitivity of 109 M. oryzae strains to Pyd was determined using mycelial growth rate assay, with EC50 values ranging from 0.291 to 2.1313 μg/mL, and an average EC50 value of 1.1005 ± 0.3727 μg/mL. Totally 28 Pyd-resistant (PydR) mutants with 15 genotypes of point mutations in succinate dehydrogenase (SDH) complex were obtained, and the resistance level could be divided into three categories of very high resistance (VHR), high resistance (HR) and moderate resistance (MR) with the resistance factors (RFs) of >1000, 105.74-986.13 and 81.92-99.48, respectively. Molecular docking revealed that all 15 mutations decreased the binding-force score for the affinity between Pyd and target subunits, which further confirmed that these 15 genotypes of point mutations were responsible for the resistance to Pyd in M. oryzae. There was positive cross resistance between Pyd and other SDHIs, such as fluxapyroxad, penflufen or carboxin, while there was no cross-resistance between Pyd and carbendazim, prochloraz or azoxystrobin in M. oryzae, however, PydR mutants with SdhBP198Q, SdhCL66F or SdhCL66R genotype were still sensitive to the other 3 SDHIs, indicating lack of cross resistance. The results of fitness study revealed that the point mutations in MoSdhB/C/D genes might reduce the hyphae growth and sporulation, but could improve the pathogenicity in M. oryzae. Taken together, the risk of resistance to Pyd might be moderate to high, and it should be used as tank-mixtures with other classes of fungicides to delay resistance development when it is used for the control of rice blast in the field.

Protein engineering often targets binding pockets or active sites which are enriched in epistasis-nonadditive interactions between amino acid substitutions-and where the combined effects of multiple single substitutions are difficult to predict. Few existing sequence-fitness datasets capture epistasis at large scale, especially for enzyme catalysis, limiting the development and assessment of model-guided enzyme engineering approaches. We present here a combinatorially complete, 160,000-variant fitness landscape across four residues in the active site of an enzyme. Assaying the native reaction of a thermostable β-subunit of tryptophan synthase (TrpB) in a nonnative environment yielded a landscape characterized by significant epistasis and many local optima. These effects prevent simulated directed evolution approaches from efficiently reaching the global optimum. There is nonetheless wide variability in the effectiveness of different directed evolution approaches, which together provide experimental benchmarks for computational and machine learning workflows. The most-fit TrpB variants contain a substitution that is nearly absent in natural TrpB sequences-a result that conservation-based predictions would not capture. Thus, although fitness prediction using evolutionary data can enrich in more-active variants, these approaches struggle to identify and differentiate among the most-active variants, even for this near-native function. Overall, this work presents a large-scale testing ground for model-guided enzyme engineering and suggests that efficient navigation of epistatic fitness landscapes can be improved by advances in both machine learning and physical modeling.