Tommie L. Lincecum

Structural and Mechanistic Basis of Pre- and Posttransfer Editing by Leucyl-tRNA Synthetase

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

Structural Dynamics of the Aminoacylation and Proofreading Functional Cycle of Bacterial Leucyl-tRNA Synthetase

Leucyl-tRNA synthetase (LeuRS) produces error-free leucyl-tRNALeu by coordinating translocation of the 3′ end of (mis-)charged tRNAs from its synthetic site to a separate proofreading site for editing. Here we report cocrystal structures of the Escherichia coli LeuRS–tRNALeu complex in the aminoacylation or editing conformations, showing that translocation involves correlated rotations of four flexibly linked LeuRS domains. This pivots the tRNA to guide its charged 3′ end from the closed aminoacylation state to the editing site. The editing domain unexpectedly stabilizes the tRNA during aminoacylation, and a large rotation of the leucine-specific domain positions the conserved KMSKS loop to bind the 3′ end of the tRNA, promoting catalysis. Our results give new insight into the structural dynamics of a molecular machine that is essential for accurate protein synthesis.

An inserted region of leucyl-tRNA synthetase plays a critical role in group I intron splicing

Yeast mitochondrial leucyl‐tRNA synthetase (LeuRS) binds to the bI4 intron and collaborates with the bI4 maturase to aid excision of the group I intron. Deletion analysis isolated the inserted LeuRS CP1 domain as a critical factor in the proteins splicing activity. Protein fragments comprised of just the LeuRS CP1 region rescued complementation of a yeast strain that expressed a splicing‐defective LeuRS. Three‐hybrid analysis determined that these CP1‐containing LeuRS fragments, ranging from 214 to 375 amino acids, bound to the bI4 intron. In each case, interactions with only the LeuRS protein fragment specifically stimulated bI4 intron splicing activity. Substitution of a homologous CP1 domain from isoleucyl‐tRNA synthetase or mutation within the LeuRS CP1 region of the smallest protein fragment abolished RNA binding and splicing activity. The CP1 domain is best known for its amino acid editing activity. However, these results suggest that elements within the LeuRS CP1 domain also play a novel role, independent of the full‐length tRNA synthetase, in binding the bI4 group I intron and facilitating its self‐splicing activity.

Cellular interactions of the cytolethal distending toxins from escherichia coli and haemophilus ducreyi

Background: Cytolethal distending toxins (CDTs) produced by pathogenic bacteria are genotoxic. Results: CDTs exploit two different endocytic pathways to reach the nucleus. Conclusion: Individual members of the CDT superfamily interact with host cells by distinct mechanisms. Significance: Learning how CDTs interact with and modulate host cells and tissues is critical for understanding the strategies used by pathogenic bacteria during infection. The cytolethal distending toxins (CDTs) compose a subclass of intracellularly acting genotoxins produced by many Gram-negative pathogenic bacteria that disrupt the normal progression of the eukaryotic cell cycle. Here, the intoxication mechanisms of CDTs from Escherichia coli (Ec-CDT) and Haemophilus ducreyi (Hd-CDT), which share limited amino acid sequence homology, were directly compared. Ec-CDT and Hd-CDT shared comparable in vitro DNase activities of the CdtB subunits, saturable cell surface binding with comparable affinities, and the requirement for an intact Golgi complex to induce cell cycle arrest. In contrast, disruption of endosome acidification blocked Hd-CDT-mediated cell cycle arrest and toxin transport to the endoplasmic reticulum and nucleus, while having no effects on Ec-CDT. Phosphorylation of the histone protein H2AX, as well as nuclear localization, was inhibited for Hd-CdtB, but not Ec-CdtB, in cells expressing dominant negative Rab7 (T22N), suggesting that Hd-CDT, but not Ec-CDT, is trafficked through late endosomal vesicles. In support of this idea, significantly more Hd-CdtB than Ec-CdtB co-localized with Rab9, which is enriched in late endosomal compartments. Competitive binding studies suggested that Ec-CDT and Hd-CDT bind to discrete cell surface determinants. These results suggest that Ec-CDT and Hd-CDT are transported within cells by distinct pathways, possibly mediated by their interaction with different receptors at the cell surface.

Characterization of benzoxaborole-based antifungal resistance mutations demonstrates that editing depends on electrostatic stabilization of the leucyl-tRNA synthetase editing cap

The broad‐spectrum benzoxaborole antifungal AN2690 blocks protein synthesis by inhibiting leucyl‐tRNA synthetase (LeuRS) via a novel oxaborole tRNA trapping mechanism in the editing site. Herein, one set of resistance mutations is at Asp487 outside the LeuRS hydrolytic editing pocket, in a region of unknown function. It is located within a eukaryote/archaea specific insert I4, which forms part of a cap over a benzoxaborole‐AMP that is bound in the LeuRS CP1 domain editing active site. Mutational and biochemical analysis at Asp487 identified a salt bridge between Asp487 and Arg316 in the hinge region of the I4 cap of yeast LeuRS that is critical for tRNA deacylation. We hypothesize that this electrostatic interaction stabilizes the cap during binding of the editing substrate for hydrolysis.