Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.

The human protein TDP-43 is a major component of the cellular aggregates found in amyotrophic lateral sclerosis and other neurodegenerative diseases. Insoluble cytoplasmic aggregates isolated from the brain of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients contain ubiquitinated, hyperphosphorylated, and N-terminally truncated TDP-43. Truncated fragments of TDP-43 identified from patient tissues contain part of the second RNA recognition motif (RRM2) and the disordered C-terminus, indicating that both domains can be involved in aggregation and toxicity. Here, we focus on RRM2. Using all-atom replica-averaged metadynamics simulations with NMR chemical shift restraints, we characterized the atomic structure of non-native states of RRM2, sparsely populated under native conditions. These structures reveal the exposure to the solvent of aggregation-prone peptide regions, normally buried in the native state, supporting a role in aggregation for the partially folded states of RRM2.

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring βα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable βα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.folding free energy surface | de novo-designed proteins | hydrogen exchange | partially folded states T he sequence space occupied by natural proteins is a small fraction of the total space accessible to a heteropolymer with 20 monomeric units (1, 2). Evolved sequences are constrained by the requirements to support ∼1,000 natural motifs (3) and to fold to a thermodynamically stable structure within seconds while maintaining biological function and avoiding aggregation. One might wonder if de novo-designed sequences not limited by history and biological constraints are capable of folding to unique, stable structures on a smooth energy surface typically observed for natural proteins. In the late 1980s, Regan and DeGrado (4) systematically aimed to synthesize a four-helix bundle protein that adopts stable folded structure in aqueous solution. Later, Harbury et al. (5) designed an unnatural right-handed tetrameric coiled coil based on design principles extracted from natural left-handed dimeric coiled coils. Subsequently, Kuhlman et al. (6) designed the sequence of a βα protein, Top7, also with a topology not found in nature. Top7 was also capable of folding to a well-defined structure (6), but its kinetic folding mechanism was complex and plagued by aggregation (7). Whether these unintended properties are a consequence of the novel topology or inherent to the sequence remains to be ...

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