Biology subjects

Risso, V. A.

Publications and source records attributed to Risso, V. A..

10 recordsLinked to original sources

bioRxiv2026

Prevalent phenotypic mutation impairs binding of broadly neutralizing antibodies to influenza hemagglutinin

Mistakes during protein synthesis, such as transcription errors, occur often and lead to non-inheritable amino acid replacements generally known as phenotypic mutations. We recently used a consensus approach in high-throughput sequencing to determine the error landscape for influenza-hemagglutinin mRNA. We found single-site errors to occur with widely different frequencies. Here we show that the most prevalent transcription error encodes a phenotypic mutation that impairs binding of broadly neutralizing antibodies. The error occurs in 0.2-0.5% of mRNA molecules and, consequently, many virions will expose hemagglutinin variants bearing the encoded amino acid replacement. Our results point to a mechanism of antibody evasion, akin to programmed recoding, in which evading mutations are encoded by transcription errors promoted by inheritable RNA sequence/structure patterns.

microbiology↗

bioRxiv2026

Influenza evolution/adaptation samples a highly non-random error landscape for hemagglutinin-encoding RNA

Natural selection acts on diversity generated by errors in the biosynthesis of the genetic material. Previous work has shown, however, that such errors are not necessarily fully random. We have used a model influenza strain and a unique-molecular-identifier-based high-throughput sequencing approach to assess the error landscape for hemagglutinin-encoding RNA. Single-site errors occur at highly variable frequencies, with differences that span several orders of magnitude, plausibly reflecting specific RNA sequence/structure patterns. Remarkably, influenza evolution/adaptation preferentially selects mutations encoded by the higher frequency errors, as shown by analyses of mutations fixed in natural strains over many decades and by analyses of antibody-escape mutations found in laboratory experiments on strains of the 2009 pandemics. Our results support that RNA error landscapes may provide information useful for predicting influenza evolution and point to high-frequency errors encoding antibody-evading mutations as potential contributors to the rapid evolution of influenza viruses.

microbiology↗

bioRxiv2025

Conformational Dynamics and Catalytic Backups in a Hyper-Thermostable Engineered Archaeal Protein Tyrosine Phosphatase

Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid-base catalysis upon loop closure. These enzymes also have a conserved phosphate binding loop that is typically highly rigid and forms a well-defined anion binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTPs high sequence similarity to its natural counterparts, ShufPTP presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active site cysteine, mechanistic promiscuity, and most notably, hyperthermostability, with a denaturation temperature likely >130 {degrees}C (>8 {degrees}C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme, and showcase the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/645524v3_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@186bfccorg.highwire.dtl.DTLVardef@19a265borg.highwire.dtl.DTLVardef@14c526corg.highwire.dtl.DTLVardef@1b75920_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Virus propagation linked to exceedingly rare gene-expression errors

Viruses are obligate parasites that establish extensive interactions with proteins and other biomolecules of their hosts. About 20% of protein molecules bear phenotypic mutations due to errors during gene expression. Phenotypic mutations are not inherited and are not purged/amplified by natural selection. Therefore, protein variants harboring phenotypic mutations remain at very low levels. Here, we show that proteins at exceedingly low levels may enable virus propagation. Bacteriophage T7 recruits the host thioredoxin as an essential processivity factor for its replisome. Thioredoxin constitutive expression yields 10000-20000 molecules per E. coli cell. We inserted early stop codons in the thioredoxin gene and appended to its end the sequence encoding for a photoconvertible fluorescent protein. Virus propagation was not abolished, indicating that some thioredoxin molecules were produced through mistranscription or mistranslation. Single-molecule localization microscopy detected 12{+/-}5 molecules per cell when an ochre codon was inserted. This work demonstrates that crucial virus-host biomolecular interactions may need occur only a few times to trigger virus propagation and supports that viruses may exploit the wide diversity of host and viral protein variants arising from gene-expression errors to establish such interactions. Immediate implications of this notion for the mechanisms of cross-species transmission and antibody evasion are discussed.

microbiology↗

bioRxiv2024

Ancestral versus Modern Substrate Scope in Family-1 Glycosidases

Experimental studies support that protein engineering based on ancestral sequence reconstruction often leads to variants with biotechnologically useful biomolecular properties. These may include high stability, enhanced conformational flexibility and a modified catalysis range. Carbohydrate-active enzymes have numerous applications related with the degradation and synthesis of carbohydrates and glycoconjugates. Here, we explore how ancestral reconstruction may impact substrate scope in glycosidases, highly diverse enzymes that catalyze the hydrolysis of glycosidic bonds in all living cells and find applications as catalysts of the reverse reaction. To this end, we screen a library of [~]500 potential glycosidase substrates for degradation by both, a modern family-1 glycosidase from Halothermothrix orenii and a putative ancestral family-1 glycosidase derived from sequence reconstruction at a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for most substrates. But the ancestral glycosidase is more efficient with flavonoid glycosides bearing large aglycon moieties. Analysis of the catalytic parameters for a selected set of substrates, alongside analysis of the library data using a supervised learning algorithm, support the hypothesis that the modern enzyme tends to become less catalytically efficient with increasing substrate size, while this trend is not observed for the ancestral glycosidase. Molecular simulations support that the ancestral catalysis pattern is linked to the existence of a highly flexible region of the ancestral structure and a cavity capable of accommodating large aglycons. Our results provide guidelines for the engineering of enzymes for the synthesis and hydrolysis of large glycoconjugates.

biochemistry↗

bioRxiv2023

Protection of catalytic cofactors by polypeptides as a driver for the emergence of primordial enzymes

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects protection of bound heme from common degradation processes and a resulting longer life time and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

molecular biology↗

bioRxiv2022

Efficient base-catalysed Kemp elimination in an engineered ancestral enzyme

The routine generation of enzymes with completely new active sites is one of the major unsolved problems in protein engineering. Advances in this field have been so far modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized, and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral {beta}-lactamase with a new active site capable of efficiently catalysing the Kemp elimination. Our Kemp eliminase is the outcome of a minimalist design based on a single function-generating mutation followed by sharply-focused, low-throughput library screening. Yet, its catalytic parameters (kcat/KM=2{middle dot}105 M-1s-1, kcat=635 s-1) compare favourably with the average modern natural enzyme and with the best proton-abstraction de novo Kemp eliminases reported in the literature. General implications of our results for de novo enzyme engineering are discussed.

biophysics↗

bioRxiv2021

Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

Obligate symbionts exhibit high evolutionary rates and extensive sequence divergence. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its refolding in vitro is very slow and its expression in E. coli leads to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro refolding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a sequence-engineering approach to rescue inefficient heterologous expression, a major biotechnological bottleneck.

biophysics↗

bioRxiv2020

Evidence for a role of phenotypic mutations in virus adaptation

Viruses interact extensively with the host molecular machinery, but the underlying mechanisms are poorly understood. Bacteriophage T7 recruits the small protein thioredoxin of the E. coli host as an essential processivity factor for the viral DNA polymerase. We challenged the phage to propagate in a host in which thioredoxin had been extensively modified to hamper its recruitment. The virus adapted to the engineered host without losing the capability to propagate in the original host, but no genetic mutations were fixed in the thioredoxin binding domain of the viral DNA polymerase. Virus adaptation correlated with mutations in the viral RNA polymerase, supporting that promiscuous thioredoxin recruitment was enabled by phenotypic mutations caused by transcription errors. These results point to a hitherto unknown mechanism of virus adaptation that may play a role in crossspecies transmission. We propose that phenotypic mutations may generally contribute to the capability of viruses to evade antiviral strategies.

evolutionary biology↗

bioRxiv2019

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionary conserved, but span at least a [~]100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding-assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.

biophysics↗