Unveiling the structural basis for translational ambiguity tolerance in a human fungal pathogen

Abstract

In a restricted group of opportunistic fungal pathogens the universal leucine CUG codon is translated both as serine (97%) and leucine (3%), challenging the concept that translational ambiguity has a negative impact in living organisms. To elucidate the molecular mechanisms underlying the in vivo tolerance to a nonconserved genetic code alteration, we have undertaken an extensive structural analysis of proteins containing CUG-encoded residues and solved the crystal structures of the two natural isoforms of Candida albicans seryl-tRNA synthetase. We show that codon reassignment resulted in a nonrandom genome-wide CUG redistribution tailored to minimize protein misfolding events induced by the large-scale leucine-to-serine replacement within the CTG clade. Leucine or serine incorporation at the CUG position in C. albicans seryl-tRNA synthetase induces only local structural changes and, although both isoforms display tRNA serylation activity, the leucine-containing isoform is more active. Similarly, codon ambiguity is predicted to shape the function of C. albicans proteins containing CUGencoded residues in functionally relevant positions, some of which have a key role in signaling cascades associated with morphological changes and pathogenesis. This study provides a first detailed analysis on natural reassignment of codon identity, unveiling a highly dynamic evolutionary pattern of thousands of fungal CUG codons to confer an optimized balance between protein structural robustness and functional plasticity.aminoacyl-tRNA synthetase | morphogenesis | mitogen-activated protein kinase pathway | Ras1 | X-ray crystallography G enetic code alterations and ambiguity are widespread in nature even though it is not yet clear how their negative impact is overcome (1). Expansion of the genetic code to selenocysteine (Sec) and pyrrolysine (Pyl) provides, however, a glimpse of advantages that may explain the evolution of codon reassignments under negative selective pressure (2). Sec is inserted into the genetic code of bacteria and eukaryotes by a specific selenocysteyl-tRNA Sec , which places selenocysteine in the catalytic site of selenoproteins increasing their chemical reaction rate relative to cysteine-containing homologues (3). Similarly, Pyl is cotranslationally introduced into the active center of methyltransferases of Methanosarcineace spp. and of Desulfitobacterium hafniense, a symbiont of the gutless worm Olavius algarvensis, where it plays a fundamental role in methane biosynthesis (2, 4). Another interesting case involves the mammalian methionyl-tRNA synthetase (MetRS), which is modified under environmental stress and misacylates noncognate tRNAs with reactive oxygen species (ROS)-scavenging methionine (5), therefore protecting proteins from oxidative damage. A similar adaptive mechanism apparently drove mitochondrial reassignment of Ile AUA codons to methionine (6).In Candida albicans and in most other CTG clade species a mutant serine tRNA (tRNA CAG Ser ) has the peculiarity of decoding leucine CUG codons both as s...

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

“…Our data are insufficient to clarify the direct involvement of this pathway in the observed resistance to the azoles; however, the nearcomplete LOH on chromosome V of the highly azole-tolerant strains (T2KO1 and T2KO2), which affected the regulatory subunit of calcineurin (CNB1) and the calcium channel (MID1), supports the hypothesis that such tolerance may be mediated through the calcineurin pathway. Furthermore, genes associated with the plasma membrane and cell wall processes are enriched in CUGs (19,24,48), suggesting that increased Leu misincorporation levels may have a stronger impact on the activation of the calcineurin pathway than on other known drug resistance pathways.…”

“…S7 and Dataset S1, Tables S10-S12). The affected region on chromosome V contains 178 ORFs (24,31). Most of these ORFs have no known function, but the known genes are involved in the regulation of biological processes (21.3%), organelle organization (13.5%), stress response (9.6%), antifungal drug resistance (6.2%), filamentous growth (9%), pathogenesis (2.8%), and conjugation (2.2%), which is in line with the phenotypic diversity observed.…”

“…Remarkably, these fungi still incorporate Ser (∼97%) and Leu (∼3%) at thousands of CUG sites present in over 60% of their genes (23). Protein X-ray crystallography and molecular modeling showed that the Leu and Ser residues encoded by CUGs are partially exposed to the solvent or on the interface of subunits of protein complexes where they can be better tolerated (24). These structural data explain the high tolerance to CUG ambiguity (23) and are fundamental to rationalize the evolution of CUG reassignment in the fungal CTG clade.…”

Seryl-tRNA Synthetases in Translation and Beyond

“…Thus, the atypical SerRS evolved a distinctive mechanism of substrate recognition. In the last few years the crystal structure of archaeal Pyrococcus horikoshii SerRS [31] (homologous to conventional, bacterial-type enzyme) has been solved, eukaryotic SerRS from fungus Candida albicans [32] and protozoa Trypanosoma brucei [33] have their structures deciphered and finally the structure of human SerRS was determined. [34] Nowadays, the complete repertoire of SerRS crystal structures is available, from all three domains of life, including lower and higher eukaryotes and different cellular compartments.…”

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

“…Indeed, fidelity can be critical for microbe and mammalian cell viability (3)(4)(5), as well as the prevention of neurological disease (5). However, in hostdependent pathogens, we and others have identified aaRS-dependent mechanisms that foster translational infidelity resulting in statistical proteins (6,7). We hypothesize that some organisms have adapted to promote or tolerate threshold levels of statistical mistranslations.…”