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Abstract: RAB27A and RAB27B are homologous small GTPases that regulate intracellular vesicle trafficking, orchestrating endocytic and exocytic processes that affect cellular communication, immune responses, and the dynamics of the cellular microenvironment. Through their interactions with effector proteins, RAB27A/B plays roles in tumor metastasis and chronic inflammation. However, pharmacological modulation of their activity faces challenges typical of small GTPases, including a lack of well-defined pockets outside the conserved GTP binding site and large RAB27-effector protein–protein interaction (PPI) surfaces. Here, we present the discovery and development of the first cell-active, rationally designed covalent inhibitors of the RAB27-effector interaction, targeting a non-conserved cysteine residue flanking the PPI interface. An electrophile-first biochemical screen led to a novel class of acrylamide covalent inhibitors, and X-ray crystallography structure-guided design led to optimized inhibitors and probes that enantioselectively target RAB27A/B Cys123 in cells. Potency and selectivity were confirmed through biochemical and cellular assays, including chemical proteomics and phenotype recapitulation in melanocytes, alongside a matched inactive enantioprobe control. In contrast, a previously reported compound, Nexinhib-20, was found to be cytotoxic and to exert its activity through non-selective reactivity. This work provides the first toolbox of cell-active chemical probes for RAB27 that can be used in future studies to shed light on the function of this protein and its potential as a therapeutic target.

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Abstract: The twin-arginine translocation (Tat) system is a mechanistically unique protein transport pathway moving folded proteins across membranes. It is found in all domains of life and is essential for bacterial virulence and plant photosynthesis. The membrane proteins, TatA, TatB and TatC form a core complex to which substrate proteins bind, triggering the recruitment of additional TatA protomers to form the transport site. Here we present cryo-electron microscopy structures of the prototypical TatBC complex from Escherichia coli and the atypical complexes from Nitratifactor salsuginis and Myxococcus xanthus in a resting state, alongside TatAC substrate-bound TatBC and TatABC complexes from E. coli in the early stages of transport. These structures demonstrate that substrate proteins associate with the core complex solely through their N-terminal signal peptides. The Tat targeting sequences of the signal peptides make specific contacts with TatC, and the peptide body is clamped by TatB. The core complex contains highly tilted transmembrane helices that drive extreme local membrane thinning. On the basis of our structures and biochemical and functional analyses, we propose a model for the early steps in Tat transport.

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Abstract: Crystallization is a key step in drug purification, offering low cost and facile scalability. The thesis investigates the role of heterogeneous nucleation templates in enhancing crystallization efficiency and controllability with a focus on biopharmaceutical applications, and examines the mechanisms of template-mediated nucleation, crystal growth, and morphology control using carbon-based templates, polymeric hydrogel templates, and microbial bio-templates. The interaction between inorganic salt and proteins was investigated and proteins themselves were also applied as the macromolecular templates. Carbon-based materials, including graphite and graphene oxide (GO), were investigated for their influence on lysozyme crystallization. Graphite reduced nucleation time by 57% compared to those without templates and demonstrated edge adsorption. GO exhibited a nonlinear effect, accelerating nucleation at low lysozyme concentrations (30 mg mL-1) while inhibiting it at higher concentrations (over 50 mg mL-1). Furthermore, a second strategy was pursued using heterogeneous templates based on poly (ethylene glycol) diacrylate (PEGDA) hydrogel microspheres (HMS). In contrast to the adsorption mechanism, the PEGDA HMS acts by releasing precipitant (0- 4 M NaCl) to create localized supersaturation gradients, thereby reducing nucleation time by 79%. Based on the mechanisms of templated crystallization observed in the lysozyme system, this work sought to explore the universality of these effects in inorganic systems critical to biomineralization and disease. The interaction between proteins and inorganic salts was further investigated in two model systems: lithium carbonate (Li'CO') and calcium oxalate (CaOx) with proteins (lysozyme, bovine haemoglobin and mRFP). In both systems, inorganic salt crystals serve as templates that influence subsequent protein adsorption and crystallization, leading to the formation of protein-salt composite crystal structures. Elevated salt concentrations consistently promoted nucleation kinetics. Proteins, however, exhibited complex effects: At low supersaturation, proteins like lysozyme inhibited Li'CO' nucleation by chelating Li'. Conversely, at high supersaturation, proteins self-assemble into oligomers or aggregates, providing additional nucleation sites and accelerating nucleation. In the CaOx system, lysozyme enhanced nucleation across its tested concentration range (0-70 mg mL-1). To bridge our findings on artificial templates to biological contexts, in vivo crystallization is further explored. Inspired by nature, the production of intracellular crystals in Bacillus thuringiensis (Bt) was studied, and its Cry1Ac gene was applied to form a crystal scaffold (CS) as the bio-template to generate crystal nanoparticles in Escherichia coli (E. coli). Through adaptive laboratory evolution (ALE) via serial passaging, we achieved a nearly tenfold increase in protein fluorescence level and produced biologically active nanocrystals with high solubility under alkaline conditions. By integrating heterogeneous nucleation theory with biomimetic strategies, our work elucidates diverse templating mechanisms, including surface, the creation of local supersaturation gradients, and inorganic salt templates. These understandings enable the rational design of templates to control crystallization outcomes. Furthermore, we establish a platform that applying the Cry gene from Bt as the crystal scaffold to function as bio-templates inside cells, demonstrating their potential in high-yield production of bioactive nanocrystals.

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Abstract: The DUF4465 family (DUF, domain of unknown function) contains more than 1000 members distributed across eight bacterial clades with species from diverse microenvironments including various gut microbiomes, hydrothermal vents, and soil. In the gut commensal Bacteroides thetaiotaomicron (B. theta), DUF4465 containing proteins act as high-affinity B12–binding proteins that scavenge this cofactor to ensure bacterial survival. Such B12 capture is essential for bacteria that have lost the ability to synthesize B12 de novo. This raises the question of whether B12-binding is ubiquitous across this family of proteins. Here, we show that B12-binding is a recurrent function of eight distantly related members of the DUF4465 family. It is reasonable to conclude that B12-binding is a common function of most DUF4465 proteins. These results establish DUF4465 as a structurally conserved family of augmented β-jellyroll B12-binding proteins with widespread roles in microbial competition for this essential cofactor.

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Abstract: Copper is an essential micronutrient for bacteria, needed for important copper enzymes such as terminal respiratory oxidases. However, in excess, copper is toxic to bacteria. This toxicity is caused by its ability to bind tightly to proteins through the formation of Cu-Cys and Cu-His bonds. To control toxicity, bacteria have evolved homeostatic systems to safely handle the copper they need while efficiently sequestering and effluxing excess copper ions. We previously found that GapA, the abundant glycolytic glyceraldehyde-3-phosphate dehydrogenase enzyme in the Staphylococcus aureus cytosol, becomes associated with copper within cells cultured in medium containing excess copper. We found that this association of GapA with copper resulted in inhibition of its enzyme activity. Here, we have characterised this binding of copper ions to S. aureus GapA in vitro to determine the mechanism of copper inhibition of GAPDH. We found that purified recombinant GapA binds a single Cu(I) ion with high affinity. Crystallographic structural determination showed association of this copper ion with two active site residues, Cys151 and His178, known to be important for catalysis. This observation was confirmed by characterisation of mutated variants lacking these residues, which showed reduced ability to bind Cu(I) ions. Finally, we demonstrated that the cytosolic copper metallochaperone, CopZ, exhibits a tighter affinity for Cu(I) and can remove copper from GapA in vitro. Together, our data demonstrate the mechanism by which excess copper binds to the S. aureus GapA enzyme and irreversibly inhibit its activity and how the cellular homeostasis system is capable of resolving this inhibition.