Biology subjects

Jackson, C. J.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Nanobodies against Plasmodium adhesins that block receptor engagement and malaria parasite invasion

Malaria is caused by Plasmodium parasites, and its clinical symptoms are a result of parasite invasion of red blood cells and the subsequent cycles of replication and proliferation. In human populations, Plasmodium vivax is responsible for the most widely distributed recurring malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity. One well-characterized family of adhesins involved in red blood cell invasion is the reticulocyte-binding-like protein homolog family, known as the RBL superfamily which includes the PfRh family in P. falciparum and PvRBP family in P. vivax. Here we report a collection of nanobodies against three members of this adhesin family, PfRh5, PfRh4 and PvRBP2b. Nanobodies against these Plasmodium adhesins bind with high affinity across several epitopes and can block receptor engagement and inhibit parasite invasion of red blood cells. Using computational design, we generated stabilized PfRh4 variants that encompass the conserved scaffold present in the PfRh and PvRBP families of adhesins and show that several variants with improved expression retained binding to mouse monoclonal antibodies, nanobodies and Complement Receptor 1, the human receptor for PfRh4. We also observed that most of the inhibitory nanobodies against the three antigens recognized the conserved structural scaffold that define this family of adhesins. These results demonstrate the potential of nanobodies to block malaria parasite invasion into red blood cells.

microbiology↗

bioRxiv2026

The modular evolution of chitinases is governed by coevolution of auxiliary and catalytic domains

The modular architecture of multi-domain enzymes is a key source of functional diversity. GH18 chitinases exemplify this, possessing a variety of auxiliary domains that have been postulated to underpin their role as essential global nutrient cyclers. However, there is no evolutionary framework that accounts for vast the assortment of domain compositions observed across the protein family. Here, we map the sequence space of nearly 40,000 bacterial chitinases, integrating phylogenetics with a quantitative analysis of domain promiscuity to decode their evolutionary history. We reveal that diversification follows distinct trajectories: enzymes necessary for environmental chitin scavenging evolve via high modular plasticity and structural elaboration of the catalytic core, whereas sequences required for specialized, essential developmental roles fix their domain architecture to fulfill lineage-specific physiological roles. By resolving how domain accretion co-evolves with catalytic scaffolds, this work provides a framework for understanding how multi-domain enzymes adapt to complex environments.

evolutionary biology↗

bioRxiv2025

Examining Selection Dynamics and Limitations in Multi-round Protein Selection of High Diversity Libraries

Proteins and peptides underpin essential biological functions and technological applications, from targeting disease-relevant interactions to providing broad enzymatic activities. However, engineering molecules with desired properties remains difficult, owing to complex sequence-structure-function relationships and the lack of data on specific systems. Experimental selection strategies, including directed evolution, phage display, and mRNA display, address this challenge by leveraging high diversity libraries and iterative enrichment under defined selection pressures. This allows for the identification of candidates without requiring extensive prior knowledge, and can generate extensive datasets for use in machine learning. While many selection systems exist, comparisons across different selection approaches are hindered by the lack of a unifying analytical framework. Here, we present a set of broadly applicable analyses for assessing selection dynamics in multi-round or multi-condition experiments, ranging from position level analysis of sequence properties to full sequence space mappings through protein language model embeddings. Using the toolset to analyze a variety of different datasets in parallel, we explore the potential effects of diversity, coverage, and reproducibility, offering generalizable insights to guide experimental design, interpretation, and troubleshooting across protein and peptide discovery platforms.

molecular biology↗

bioRxiv2025

Fragment Screening and Structure-Guided Development of Heparanase Inhibitors Reveals Orthosteric and Allosteric Inhibition

Heparanase is the sole enzyme responsible for breaking down heparan sulfate within the extracellular matrix and its overexpression is linked to human diseases. Despite heparanase being a promising drug target, most efforts have focused on substrate mimetics, which have failed clinical trials, highlighting the need for new inhibitor scaffolds. Here, we employed fragment-based drug design to explore novel chemical space to develop small molecule inhibitors of heparanase. We used a crystallographic and computational approach to identify 31 fragments that bind heparanase; five of these inhibited heparanase in the micromolar range. One of these fragments underwent two cycles of fragment growing, which resulted in a compound with a seven-fold increased potency compared to the initial hit. The results from our fragment screen unveil untapped chemical space for heparanase inhibition, paving the way for the development of potent drug leads with the potential to transform the treatment of heparanase-related diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/679132v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@4a5c9corg.highwire.dtl.DTLVardef@61340org.highwire.dtl.DTLVardef@d9d2ddorg.highwire.dtl.DTLVardef@a2cf82_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Structural and Evolutionary Constraints of Organophosphate Resistance in Dipteran Carboxylesterases

Enzymatic detoxification of organophosphate (OP) insecticides can confer resistance in some insects, yet the precise molecular basis of this trait, and how it has evolved, remains poorly understood. In certain dipteran species, a G[->]D mutation in the oxyanion hole of -carboxylesterases (CBEs) enhances OP hydrolysis, yet this adaptation is not widespread despite the presence of orthologous CBEs in other insect species that are also exposed to OPs. The extent, and molecular basis, of evolutionary contingency and epistasis in this catalytic OP resistance has not been explored, and how further mutations might optimize OP detoxification in the future is not clear. Here, we systematically compare OP hydrolysis and analyse structures of CBE orthologs across several dipteran species, revealing that the success of the G137D mutation is sequence context-dependent. We employed laboratory-directed evolution to enhance OP turnover over 1000-fold vs. the wild-type enzyme and tested these variants in transgenic Drosophila melanogaster, demonstrating that improved catalytic rates do not directly translate to increased resistance. By highlighting the trade-off between organophosphate affinity and turnover, this work further clarifies the complex evolutionary trajectories determining why a particular resistance mechanism may evolve in some species but not others. SignificanceThis study reveals the intricate evolutionary path to insecticide resistance in insects, highlighting why a potent resistance mutation is effective in some species but not others. We show that the mutations success is contingent on the enzymes pre-existing structural features, highlighting the strong intramolecular epistasis. Using laboratory evolution, we enhanced the enzymes detoxification activity over 1000-fold, yet discovered this did not translate to increased resistance in transgenic flies. This surprising result demonstrates that effective real-world resistance requires a delicate balance between an enzymes ability to bind an insecticide (affinity) and its speed at breaking it down (turnover), providing crucial insights into the constraints governing molecular adaptation.

biochemistry↗

bioRxiv2025

Single-Molecule Kinetic Exploration of Functional Substates in an Evolving Phosphotriesterase

Enzymes achieve catalysis by dynamically sampling diverse conformational states. Beyond this plasticity, individual enzyme molecules occupy metastable substates, forming an ensemble of functional substates within a population. Since shifts in functional substate dynamics drive phenotypic variation, their evolutionary trajectories are central to the emergence of new functions. However, the challenge of measuring functional substates has hindered our understanding of their role in enzyme evolution and the optimization of conformational substates. Here, we address this gap by investigating how functional and conformational substates were modulated during enzyme functional transitions, using single-molecule kinetic assays and molecular dynamics simulations. We analyzed wild-type phosphotriesterase (PTE) and 18 evolved variants that transitioned from the native PTE to promiscuous arylesterase (AE) activity. Our findings reveal that evolutionary transitions reshape functional and conformational substate landscapes: PTE-specialized variants exhibit broader substate distributions, whereas AE-specialized variants display more uniform substates. These results provide the first direct evidence that enzyme evolution is accompanied by coordinated shifts in functional and conformational substate equilibria, optimizing both for the enzymes catalytic efficiency. This work highlights the power of single-molecule techniques in uncovering how heterogeneous enzyme populations navigate substate transitions and, ultimately, how these transitions shape enzyme evolvability.

biochemistry↗

bioRxiv2025

Fitness Landscape Ruggedness Arises from Biophysical Complexity

The ruggedness of a proteins fitness landscape constrains how it can evolve and how effectively it can be engineered. What determines whether a fitness landscape is rugged, and where in sequence space that ruggedness concentrates, remains poorly understood. Existing measures of ruggedness rely on complete combinatorial datasets and cannot be applied to landscapes in which only a fraction of possible sequences have been characterized. Here, we introduce a graph-based measure of ruggedness based on how quickly fitness differences diffuse across a landscape, which can be applied to sparse datasets. Applying this measure to 64 protein domains and several experimental systems with characterized folding and binding constraints, we show that ruggedness is not uniformly distributed but concentrates at regions where the dominant constraint on fitness changes. These results establish that the composition of individually simple constraints can generate localized ruggedness, providing a mechanistic basis for the complexity of protein fitness landscapes.

evolutionary biology↗

bioRxiv2024

Exploring large protein sequence space through homology- and representation-based hierarchical clustering

Exploration of protein sequence space can offer insight into protein sequence-function relationships, benefitting both basic science and industrial applications. The use of sequence similarity networks (SSNs) is a standard method for exploring large sequence datasets, but is currently limited when scaling to very large datasets and when viewing more than one level (hierarchy) of homology. Here, we present a sequence analysis pipeline with a number of innovations that address some limitations of traditional SSNs. First, we develop a hierarchical visualization approach that captures the full range of homologies across protein superfamilies. Second, we leverage representations embedded by protein language models as an alternative homology metric to the basic local alignment search tool (BLAST), showing that they produce comparable results when identifying isofunctional protein families. Finally, we demonstrate that unbiased representative sampling of sequences from genetic neighborhoods can be achieved through the use of hidden Markov models (HMMs) or vector representations. The utility of these methods is exemplified by updating the sequence-function analysis of the FMN/F420-binding split barrel superfamily and improving phylogenetic analyses. We provide our sequence exploration pipeline as publicly available code (ProteinClusterTools) and show it to be scalable to large datasets ([~]300k sequences) using desktop computers.

bioinformatics↗

bioRxiv2024

Structure, dynamics and evolution of the Candida albicans multi-drug resistance ABC transporter CDR1

The pleiotropic drug resistance transporter Cdr1 from Candida albicans plays a crucial role in antifungal resistance. Here, we present high-resolution cryo-electron microscopy structures of C. albicans Cdr1 in multiple functional states: nucleotide-bound, substrate-bound, and apo forms. The 3.5 [A] resolution structure of Cdr1 in complex with ATP and ADP reveals the molecular details of its asymmetric nucleotide-binding sites (NBS), with ATP bound to the deviant NBS1 and ADP to the canonical NBS2. Structures of Cdr1 bound to rhodamine 6G (3.4 [A]) and Oregon Green 488 (3.5 [A]) in complex with these nucleotides elucidate the pleiotropic substrate-binding pocket and highlight how nucleotide exchange drives conformational change required for transport. Additionally, we determined a 3.7 [A] resolution structure of Cdr1 in the detergent LMNG without nucleotides, as well as a 3.5 [A] resolution structure with nucleotides but no substrate, representing an apo state. We complemented these structural insights with molecular dynamics simulations to understand substrate binding dynamics, ancestral sequence reconstruction to trace the evolution of key functional motifs, and analysis of clinical isolates from sequence databases to identify potential resistance-associated variations. Comparison of these structures provides new insights into the conformational changes associated with the transport cycle of this asymmetric ABC transporter, revealing how ATP binding at the deviant NBS1 allosterically regulates the canonical NBS2, driving ATPase activity. This work significantly advances our understanding of the molecular mechanisms underlying multidrug resistance in pathogenic fungi and provides a structural and evolutionary framework for the rational design of Cdr1 inhibitors to combat antifungal resistance.

microbiology↗

bioRxiv2024

Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 4-Cyanotryptophan Conserves Their Structure and Enables the Detection of Ligand Binding Sites

Cyano-tryptophans (CN-Trp) are privileged multimodal reporters on protein structure. They are similar in size to the canonical amino acid tryptophan and some of them exhibit bright fluorescence which responds sensitively to changes in the environment. We selected aminoacyl-tRNA synthetases specific for 4-, 5-, 6-, and 7-CN-Trp for high-yield in vivo production of proteins with a single, site-specifically introduced nitrile label. The absorption maximum of 4-CN-Trp is distinct from Trp, allowing the selective excitation of its intense fluorescence. 4-CN-Trp features bright fluorescence in the visible range. Crystal structures of maltose binding protein demonstrate near-complete structural conservation when a native buried Trp residue is replaced by 4-CN-Trp. Besides presenting an inconspicuous tag for live cell microscopy, the high fluorescence of 4-CN-Trp enables measurements of subnanomolar ligand binding affinities in isotropic solution, as demonstrated by the complex between rapamycin and the peptidyl-prolyl isomerase FKBP12 furnished with a 4-CN-Trp residue in the substrate binding pocket. Furthermore, 4-CN-Trp residues positioned at different locations of a protein containing multiple tryptophan residues permits using fluorescence quenching experiments to detect the proximity of individual Trp residues to the binding site of aromatic ligands.

biochemistry↗

bioRxiv2024

Isolation, identification, and characterisation of the malachite green detoxifying bacterial strain Bacillus pacificus ROC1 and the azoreductase AzrC

Malachite green (MG) is used as a dye for materials such as wood, cotton, and nylon, and is used in aquaculture to prevent fungal and protozoan diseases. However, it is highly toxic, with carcinogenic, mutagenic, and teratogenic properties, resulting in bans worldwide. Despite this, MG is still frequently used in many countries due to its efficacy and economy. MG is persistent in the environment and so requires degradative intervention. In this work we isolated Bacillus pacificus ROC1 strain from a salt flat in Pakistan that had the ability to aerobically detoxify MG, as determined by bacterio- and phyto-toxicity assays. We demonstrate immobilized B. pacificus ROC1 can effectively detoxify MG, which highlights a potential method for its biodegradation. Genomic sequencing identified three candidate azo-reductases within B. pacificus ROC1 that could be responsible for the MG-degrading activity. These were cloned, expressed and purified from Escherichia coli, with one (AzrC), catalyzing the reduction of MG to leuco-MG in vitro. AzrC was crystallised and MG was captured within the active site in a Michaelis complex, providing structural insight into the reduction mechanism. Altogether, this work identifies a bacterium capable of aerobically degrading a major industrial pollutant and characterizes the molecular basis for this activity.

biochemistry↗

bioRxiv2024

Nitration of a ribosomal pentapeptide generates a noncanonical precursor for nonribosomal peptide synthesis

Peptide natural products possess a fascinating array of complex structures and diverse functions. Central to this is a repertoire of modified amino acid building blocks, which stem from fundamentally different biosynthesis pathways for peptides of nonribosomal and ribosomal origins. Given these origins, integration of nonribosomal and ribosomal pathways have previously been thought unlikely. Now, we demonstrate that ribosomal biosynthesis generates a key noncanonical 3-nitrotyrosine building block for the nonribosomal synthesis of rufomycin. In this pathway, a biarylitide-type ribosomal peptide is nitrated by a modified cytochrome P450 crosslinking enzyme, with the nitrated residue liberated by the actions of a dedicated protease found within the rufomycin gene cluster before being incorporated into rufomycin by the rufomycin nonribosomal peptide synthetase. This resolves the enigmatic origins of 3-nitrotyrosine within rufomycin biosynthesis and demonstrates unexpected integration of ribosomal peptide synthesis as a mechanism for the generation of noncanonical building blocks within nonribosomal synthesis pathways.

biochemistry↗

bioRxiv2024

Ancestral reconstruction of polyethylene terephthalate degrading cutinases reveals a rugged and unexplored sequence-fitness landscape

The enzymatic degradation of polyethylene terephthalate (PET) is a promising method of advanced plastic recycling. Traditional protein engineering methods often fall short in exploring protein sequence space for optimal enzymes due to structural and rational design limitations. Our study addresses this by using multiplexed ancestral sequence reconstruction (mASR) to explore the evolutionary sequence space of PET-degrading cutinases. With a dataset of 397 cutinase sequences, we created a diverse library of ancestral sequences. Experimental characterization of 48 ancestral sequences revealed a wide range of PETase activities, highlighting the value of mASR in uncovering functional variants when compared to traditional ASR. Our results showed that PETase activity in cutinases evolved through diverse pathways involving distal mutations to the active site, and is readily accessible within this family. Additionally, our analysis of the PETase fitness landscape using one-hot encoding (OHE) and local ancestral sequence embedding (LASE) highlighted the effectiveness of LASE in capturing sequence features relevant to activity. This work emphasizes the utility of mASR as a protein engineering tool for identifying enhanced PET-degrading enzymes, and the advantages of the LASE embedding scheme in mapping the PETase fitness landscape.

biochemistry↗

bioRxiv2023

Leveraging ancestral sequence reconstruction for protein representation learning

Protein language models (PLMs) convert amino acid sequences into the numerical representations required to train machine learning (ML) models. Many PLMs are large (>600 M parameters) and trained on a broad span of protein sequence space. However, these models have limitations in terms of predictive accuracy and computational cost. Here, we use multiplexed Ancestral Sequence Reconstruction (mASR) to generate small but focused functional protein sequence datasets for PLM training. Compared to large PLMs, this local ancestral sequence embedding (LASE) produces representations 10-fold faster and with higher predictive accuracy. We show that due to the evolutionary nature of the ASR data, LASE produces smoother fitness landscapes in which protein variants that are closer in fitness value become numerically closer in representation space. This work contributes to the implementation of ML-based protein design in real-world settings, where data is sparse and computational resources are limited.

bioinformatics↗

bioRxiv2023

Origin Of Evolutionary Bifurcation In An Enzyme

Evolution can lead to significantly distinct outcomes depending on the mutational path taken. Evolutionary bifurcation, in which two mutational trajectories segregate, becoming non-interchangeable over time, is the basis of diversification in all kingdoms of life. Here, we present a detailed molecular description of a bifurcation event that rapidly led to the emergence of two distinct enzymes from a common ancestor. When initiated from two starting points that differed by a single amino acid, the laboratory evolution of a phosphotriesterase (PTE) toward arylester hydrolysis resulted in different genetic and phenotypic outcomes. One trajectory led to a >35,000-fold increase in activity via the reorganization of its active site to achieve exquisite enzyme-substrate complementarity. The second trajectory gave rise to an evolved variant with a [~]500-fold increase in activity, but exhibiting an alternative substrate binding mode resulting from the destabilization of an active site loop. While initial mutations tend to dictate mutational accessibility, we rather observed the gradual divergence and specialisation of each trajectory, following the emergence of distinct molecular interaction networks. Intramolecular epistasis underlay pathway bifurcation by promoting unique synergistic interactions within each trajectory, while restricting the fixation of mutation across pathways. Our results illustrate how distinct molecular outcomes can radiate from a common protein ancestor and give rise to phenotypic diversity.

evolutionary biology↗

bioRxiv2023

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally significant proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in [a]-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present the first structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate (RuBP), and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial [a]-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of -carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. One-Sentence SummaryThe carboxysomal carbonic anhydrase, CsoSCA, is allosterically activated by the Rubisco substrate RuBP, revealing a novel mechanism controlling key enzyme activity in cyanobacterial -carboxysomes.

molecular biology↗

bioRxiv2023

A marker chromosome in psychosis identifies glycine decarboxylase (GLDC) as a novel regulator of neuronal and synaptic function in the hippocampus

The biological significance of a small supernumerary marker chromosome that results in dosage alterations to chromosome 9p24.1, including triplication of the GLDC gene encoding glycine decarboxylase, in two patients with psychosis is unclear. In an allelic series of copy number variant mouse models, we identify that triplication of Gldc reduces extracellular glycine levels as determined by optical fluorescence resonance energy transfer (FRET) in dentate gyrus (DG) but not in CA1, suppresses long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduces the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displays deficits in prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference. Our results thus provide a link between a genomic copy number variation, biochemical, cellular and behavioral phenotypes, and further demonstrate that GLDC negatively regulates long-term synaptic plasticity at specific hippocampal synapses, possibly contributing to the development of neuropsychiatric disorders.

neuroscience↗

bioRxiv2023

Active site mutations of F420-dependent alkene reductases reverse stereoselectivity

Ene-reductases from the Flavin/Deazaflavin Oxidoreductase (FDOR) family have potential value in biocatalysis as they typically exhibit complementary stereoselectivity to the widely utilized Old Yellow Enzyme (OYE) family, yet they are comparatively poorly understood at a mechanistic level. Here, we use a rational design approach to generate a library of 46 active site mutants of two FDORs from Mycobacterium smegmatis and examine the effects on conversion and stereoselectivity against a panel of substrates. Analysis of the effects of these mutations on stereoselectivity across all substrates revealed that the catalytic mechanism is highly sensitive to the polarity of the immediate active site. A conserved active site tyrosine in these enzymes, which does not serve as the proton donor, strongly affects stereochemical outcomes with C- (but not C{beta}-) substituted substrates. Notably, a Tyr-Met mutation at this position reversed the diastereomeric excess (de) with (R)-carvone from 85.3% to -17.3% (cis/trans). Additionally, this mutation significantly increases activity with (1S)- verbenone. Finally, we show that the altered stereoselectivity is not due to a "flipped" substrate binding mode in these mutants, but rather that the hydrogenation mode is altered to favor syn relative to anti addition. These results show that the FDORs are highly engineerable and that, despite their superficial similarity, the OYE and FDOR families differ in crucial mechanistic aspects.

synthetic biology↗