Biology subjects

Boucher, J. I.

Publications and source records attributed to Boucher, J. I..

4 recordsLinked to original sources

bioRxiv2025

Mutational scan of self-cleavage by HIV-1 protease provides new views of a conformationally dynamic mechanism

The mechanism of conformationally dynamic proteins remains understudied because they are difficult to analyze structurally. For HIV-1 protease the mechanism of cleavage by mature protease is well understood in large part because it forms a stable structure that is amenable to x-ray crystallography. However, self-cleavage or autoproteolysis of protease from the viral polyprotein involves transiently populated structures and is poorly understood. We probed autoproteolysis in HIV-1 using a yeast reporter and mutational scanning. We compared our results with mutational scanning of protease on viral fitness, which integrates both autocleavage and cutting by mature enzyme. We identified 220 mutations that were well tolerated for self-cleavage but not fitness. We analyzed three of these mutations (D30E, W42M and P44L) using independent approaches. All three were capable of efficient self-cleavage in a bacterial assay of autoproteolysis, but had strong defects in mature form for cleavage of a peptide substrate. These separation of function mutations from the mutational scan clustered at hot-spot locations that do not impact autoproteolysis likely because they are conformationally dynamic during self-cleavage. We used the mutational scanning results and molecular simulations to provide models of autoproteolysis conformations. These models provide new views of a structurally dynamic mechanism.

biochemistry↗

bioRxiv2023

Strong epistasis constrains the evolution of strict substrate specificity in apicomplexan lactate dehydrogenases

The homologous enzymes lactate and malate dehydrogenase (L/MDH) are structurally similar but are specific for different substrates. LDH vs MDH specificity is canonically governed by the identity of a single "specificity residue" at position 102. However, LDH function has convergently evolved from a specific MDH at least four times, and the catalytic role of residue 102 is not conserved between different phyla. The apicomplexa are a phylum of obligate, intracellular eukaryotic parasites responsible for wide-spread disease such as Plasmodium falciparum (malaria), Cryptosporidium parvum (cryptosporidiosis), Toxoplasma gondii (toxoplasmosis), and Eimeria maxima (eimeriosis). The apicomplexan LDH evolved via a five-residue insertion that produced a novel specificity residue, W107f. The commonly accepted mechanism of LDH specificity involves charge balance and steric occlusion, but our data shows that the general mechanism of apicomplexan LDHs does not use W107f as a steric block. Only Plasmodium LDHs evolved substantial steric specificity, making them exceptional among Apicomplexa. Strong protein epistasis constrained this evolution, making it difficult to revert to ancestral phenotypes. Here, we use ancestral sequence reconstruction (ASR), steady-state kinetics, and x-ray crystallography to characterize apicomplexan LDHs which challenge current assumptions about the evolution of L/MDH activity. We demonstrate the unique specificity of Plasmodium LDHs and identify the active site residues controlling their substrate recognition. The extraordinarily high specificity of Plasmodium LDHs presents difficulties for small-molecule inhibitor development, and successful drugs against Plasmodium LDH may not be efficacious against other Apicomplexa LDHs and their diseases.

evolutionary biology↗

bioRxiv2018

Functional and structural resilience of the active site loop in the evolution of Plasmodium lactate dehydrogenase.

The malarial pathogen Plasmodium falciparum (Pf) is a member of the Apicomplexa, which independently evolved a highly specific lactate dehydrogenase (LDH) from an ancestral malate dehydrogenase (MDH) via a five-residue insertion in a key active site loop. PfLDH is widely considered an attractive drug target due to its unique active site. Apicomplexan loop conservation suggests that a particular insertion sequence was required to evolve LDH specificity, and we previously showed (Boucher 2014) that a tryptophan in the insertion, W107f, is essential for activity and specificity. However, the roles of other residues in the loop are currently unknown. Here we show that PfLDH activity is remarkably resilient to radical perturbations of both loop identity and length. Thus, alternative insertions could have evolved LDH specificity as long as they contained a tryptophan in the proper location. PfLDH therefore has high potential to develop resistance to drugs that target its distinctive active site.

biochemistry↗

bioRxiv2018

Constrained mutational sampling of amino acids in HIV-1 protease evolution

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution are interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available dataset of 32,163 sequenced isolates from drug-naive individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple base mutations in HIV-1 protease is strongly influenced by mutational sampling.

evolutionary biology↗