Biology subjects

Jackson, C. J.

Publications and source records attributed to Jackson, C. J..

4 recordsLinked to original sources

bioRxiv2017

Cryptic genetic variation defines the adaptive evolutionary potential of enzymes

Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may also impact the evolutionary potential of proteins, but the molecular basis for this remains unclear. Here we perform comparative directed evolution in which four orthologous metallo-{beta}-lactamases were evolved toward a new function. We found that genetic variation between these enzymes resulted in distinct evolutionary outcomes. The ortholog with the lower initial activity reached a 20-fold higher fitness plateau exclusively via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show that the cryptic molecular properties and conformational variation of residues in the initial genotypes cause epistasis, thereby constraining evolutionary outcomes. Our work highlights that understanding the molecular details relating genetic variation to protein functions is essential to predicting the evolution of proteins.

evolutionary biology↗

bioRxiv2017

Overcoming insecticide resistance through computational inhibitor design

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could therefore restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent computational design to produce nano/pico-molar boronic acid inhibitors of the carboxylesterase E7 from the agricultural pest Lucilia cuprina, as well as a common Gly137Asp E7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase, and low to no toxicity in human cells and mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

biochemistry↗

bioRxiv2017

Evolution of an enzyme from a solute-binding protein

Much of the functional diversity observed in modern enzyme superfamilies originates from molecular tinkering with existing enzymes1. New enzymes frequently evolve from enzymes with latent, promiscuous activities2, and often inherit key features of the ancestral enzyme, retaining conserved catalytic groups and stabilizing analogous intermediates or transition states3. While experimental evolutionary biochemistry has yielded considerable insight into the evolution of new enzymes from existing enzymes4, the emergence of catalytic activity de novo remains poorly understood. Although certain enzymes are thought to have evolved from non-catalytic proteins5-7, the mechanisms underlying these complete evolutionary transitions have not been described. Here we show how the enzyme cyclohexadienyl dehydratase (CDT) evolved from a cationic amino acid-binding protein belonging to the solute-binding protein (SBP) superfamily. Analysis of the evolutionary trajectory between reconstructed ancestors and extant proteins showed that the emergence and optimization of catalytic activity involved several distinct processes. The emergence of CDT activity was potentiated by the incorporation of a desolvated general acid into the ancestral binding site, which provided an intrinsically reactive catalytic motif, and reshaping of the ancestral binding site, which facilitated enzyme-substrate complementarity. Catalytic activity was subsequently gained via the introduction of hydrogen-bonding networks that positioned the catalytic residue precisely and contributed to transition state stabilization. Finally, catalytic activity was enhanced by remote substitutions that refined the active site structure and reduced sampling of non-catalytic states. Our work shows that the evolutionary processes that underlie the emergence of enzymes by natural selection in the wild are mirrored by recent examples of computational design and directed evolution of enzymes in the laboratory.

biochemistry↗

bioRxiv2017

Multivalency drives the neutralizing activity of antibodies against the Plasmodium falciparum circumsporozoite protein

The repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.\n\nAuthor SummaryVaccines aim to protect by inducing the immune system to make molecules called antibodies that can recognize molecules on the surface of invading pathogens. In the case of malaria, our most advanced vaccine candidates aim to promote the production of antibodies that recognize the circumsporozoite protein (CSP) molecule on the surface of the invasive parasite stage called the sporozoite. In this report we use X-ray crystallography to determine the structure of CSP-binding antibodies at the atomic level. We use other techniques such as isothermal titration calorimetry and structural modeling to examine how this antibody interacts with the CSP molecule. Strikingly, we found that each CSP molecule could bind 6 antibodies. This finding has implications for the immune response and may explain why high titers of antibody are needed for protection. Moreover, because the structure of the CSP repeat is quite simple we determined that the number of different kinds of antibodies that could bind this molecule are quite small. However a high avidity interaction between those antibodies and CSP can result from a process called affinity maturation that allows the body to learn how to make improved antibodies specific for pathogen molecules. These data show that while it is challenging for the immune system to recognize and neutralize CSP, it should be possible to generate viable vaccines targeting this molecule.

immunology↗