ABSTRACT
A bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38â Å. Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the spaces that accommodate the substituents attached to the P atom of several products of organophosphate substrates after detachment of the leaving group. The crystal structures of the complexes of phosphotriesterase with the organophosphate products reveal similar short interaction distances of the two partially charged O atoms of the P-O bonds with the exposed ß-Zn2+ ion and the buried α-Zn2+ ion. This suggests that both Zn2+ ions have a role in stabilizing the transition state for substrate hydrolysis. Overall, this study provides valuable insights into the challenges and considerations involved in studying the crystal structures of ligand-protein complexes, highlighting the importance of careful experimental design and rigorous data analysis in ensuring the accuracy and reliability of the resulting phosphotriesterase-organophosphate structures.
Subject(s)
Phosphoric Triester Hydrolases , Phosphoric Triester Hydrolases/chemistry , Phosphoric Triester Hydrolases/metabolism , Crystallization , Ligands , Reproducibility of Results , Organophosphates , Crystallography, X-RayABSTRACT
Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.
Subject(s)
Recombinant Proteins , Serum Albumin , Animals , Humans , Disulfides , Escherichia coli/genetics , Reproducibility of Results , Serum Albumin/genetics , Serum Albumin/chemistry , Serum Albumin, Human/chemistry , Serum Albumin, Human/genetics , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.
Subject(s)
Cell Membrane/drug effects , Drosophila Proteins/antagonists & inhibitors , Ion Channel Gating/drug effects , Kv1.2 Potassium Channel/antagonists & inhibitors , Mollusk Venoms/toxicity , Shaker Superfamily of Potassium Channels/antagonists & inhibitors , Animals , Cell Membrane/metabolism , Crystallography, X-Ray , Drosophila Proteins/genetics , Drosophila Proteins/isolation & purification , Drosophila Proteins/metabolism , Drug Design , Female , Hydrogen Bonding/drug effects , Kv1.2 Potassium Channel/genetics , Kv1.2 Potassium Channel/isolation & purification , Kv1.2 Potassium Channel/metabolism , Lethal Dose 50 , Molecular Docking Simulation , Molecular Dynamics Simulation , Mollusk Venoms/chemistry , Mutation , Oocytes , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Shaker Superfamily of Potassium Channels/genetics , Shaker Superfamily of Potassium Channels/isolation & purification , Shaker Superfamily of Potassium Channels/metabolism , Water/chemistry , Water/metabolism , Xenopus laevisABSTRACT
Proteins have evolved to balance efficient binding of desired partners with rejection of unwanted interactions. To investigate the evolution of protein-protein interactions, we selected a random library of pre-stabilized TEM1 ß-lactamase against wild-type TEM1 using yeast surface display. Three mutations were sufficient to achieve micromolar affinity binding between the two. The X-ray structure emphasized that the main contribution of the selected mutations was to modify the protein fold, specifically removing the N'-terminal helix, which consequently allowed protein coupling via a ß-sheet-mediated interaction resembling amyloid interaction mode. The only selected mutation located at the interaction interface (E58V) is reminiscent of the single mutation commonly causing sickle-cell anemia. Interestingly, the evolved mutations cannot be inserted into the wild-type protein due to reduced thermal stability of the resulting mutant protein. These results reveal a simple mechanism by which undesirable binding is purged by loss of thermal stability.