Biology subjects

Campopiano, D. J.

Publications and source records attributed to Campopiano, D. J..

3 recordsLinked to original sources

bioRxiv2025

De novo acyl carrier proteins display structure-independent modification and sequence novelty

Acyl carrier proteins (ACPs) are dynamic, structurally conserved -helical proteins central to many primary and secondary metabolic processes. Whilst prior engineering efforts have focused on strategic mutagenesis and "helix swaps", much of the ACP sequence design space remains underexplored. Here, we create diverse variants of the archetypal ACP subclass - AcpP - using a bespoke sequence-generating algorithm (ALGO-CP), which utilises a combined evolutionary and physicochemical design approach. Using ALGO-CP, we generated two soluble candidates - ALGO-055 and ALGO-059 - that can undergo full post-translational modification from apo[->]holo[->]acyl forms in vitro, using recombinant modifying enzymes. Building on these successful designs, we further adapted ALGO-CP to produce several chimeras, two of which - chALGO-012 and chALGO-024 - also exhibit full modifiability. We explore the structural plasticity of our ALGO variants via robust molecular dynamic simulations, and we further reveal by circular dichroism spectroscopy that ALGO-055 and ALGO-059 lack the canonical -helical fold of an ACP, whilst remaining soluble and readily modifiable. Upon acylation of ALGO-055 and ALGO-059, we observe a marked increase in helicity indicative of partial restructuring. Of note, both ALGO-055 and ALGO-059 harbour several rare amino acid variations across their sequences, whilst preserving many important acidic "hotspots" involved in key protein-protein interactions. By testing the limits of the AcpP design space, our findings suggest that some key aspects of ACP behaviour (specifically post-translational modification) can be retained independently of the canonical structure. This work establishes a foundation for probing ACP sequence diversity through a hybrid computational-experimental approach. ALGO-CP is available under AGPL3.0 license: https://github.com/MAHerrera-94/ALGO_CP.

bioengineering↗

bioRxiv2022

Designing human Sphingosine-1-phosphate lyases using a temporal Dirichlet variational autoencoder

Enzymatic deficiencies cause the accumulation of toxic levels of substrates in a cell and are associated with life-threatening pathologies. Restoring physiological enzymes levels by injecting a recombinant version of the defective enzyme could provide a viable therapeutic option. However, these enzyme replacement therapies have had limited success, as the recombinant enzymes are less catalytically active, cause immune response and are difficult to manufacture. Moreover, the vast sequence design space makes finding enzymes with desired therapeutic properties extremely challenging. Here, we present a new enzyme engineering framework, which builds on recent advances in deep learning, variational calculus and natural language processing, to design variants of human enzymes with biochemical features comparable to the wild type protein as a way to rapidly build targeted libraries for downstream screening. We applied our method to design variants of human Sphyngosine-1-phosphate lyase (HsS1PL) as potential therapeutic treatments for nephrotic syndrome type 14 (NPHS14), and characterized their biochemical properties through extensive sequence and molecular dynamics analyses.

bioinformatics↗

bioRxiv2021

Inositol lipid synthesis is widespread in host-associated Bacteroidetes

Ubiquitous in eukaryotes, inositol lipids have finely tuned roles in cellular signaling and membrane homeostasis. In Bacteria, however, inositol lipid production is rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids, including inositol sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, we investigated the gene cluster responsible for inositol lipid synthesis in BT using a novel strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol-dihydroceramide, including structural and kinetic studies of the enzyme MIP synthase (MIPS). We determined the crystal structure of recombinant BT MIPS with bound NAD cofactor at 2.0 [A] resolution, and identified the first reported phosphatase for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP) to phosphatidylinositol (PI). Transcriptomic analysis indicated inositol production is nonessential but its loss alters BT capsule expression. Bioinformatic and lipidomic comparisons of Bacteroidetes species revealed a novel second putative pathway for bacterial PI synthesis without a PIP intermediate. Our results indicate that inositol sphingolipid production, via one of the two pathways, is widespread in host-associated Bacteroidetes, and may be implicated in host interactions both indirectly via the capsule and directly through inositol lipid provisioning.

microbiology↗