Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase.

D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD's invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD's chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2'-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD's activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor-binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with stabilized Spike ectodomain. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high-affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high-affinity (0.53-4.2 nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron and Delta pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

A discriminator code-based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria.

D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, attached to tRNA. We have recently shown that this activity enables DTD to clear non-cognate Gly-tRNAAla with 1000-fold higher efficiency than its activity on Gly-tRNAGly, indicating tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's discriminator base predominantly accounts for this activity difference and is the key to selection by DTD. Accordingly, the uracil discriminator base, serving as a negative determinant, prevents Gly-tRNAGly misediting by DTD and this protection is augmented by EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only G3•U70 for tRNAGly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine misincorporation in proteins rather than only recycling mischarged tRNAAla. Overall, the study reveals the unique co-evolution of DTD and discriminator base, and suggests DTD's strong selection pressure on bacterial tRNAGlys to retain a pyrimidine discriminator code.

Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS.

Strict L-chiral rejection through Gly-cisPro motif during chiral proofreading underlies the inability of D-aminoacyl-tRNA deacylase (DTD) to discriminate between D-amino acids and achiral glycine. The consequent Gly-tRNAGly 'misediting paradox' is resolved by EF-Tu in the cell. Here, we show that DTD's active site architecture can efficiently edit mischarged Gly-tRNAAla species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error. Also, DTD knockout in AlaRS editing-defective background causes pronounced toxicity in Escherichia coli even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNAAla-specific G3•U70. Moreover, DTD's activity on non-cognate Gly-tRNAAla is conserved across all bacteria and eukaryotes, suggesting DTD's key cellular role as a glycine deacylator. Our study thus reveals a hitherto unknown function of DTD in cracking the universal mechanistic dilemma encountered by AlaRS, and its physiological importance.