Biology subjects

Sussman, J. L.

Publications and source records attributed to Sussman, J. L..

4 recordsLinked to original sources

bioRxiv2023

The impact of molecular variants, crystallization conditions and space group on structure-ligand complexes: A case study on Bacterial Phosphotriesterase Variants and complexes

While attempting to study the 3D structure of proteins with bound ligands, one often encounters considerable difficulties. We illustrate, as an example, the bacterial enzyme phosphotriesterase and specifically examine the effects of multiple factors such as the molecular constructs, ligands used during protein expression and purification, crystallization precipitance, and space group on the visualization of molecular complexes of organophosphate ligands bound to the enzyme. We analyzed twelve crystal structures of the different phosphotriesterase constructs derived by directed evolution in both apo and holo forms (in complex with organophosphate analogs), with resolutions up to 1.38 [A]. 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 molecular complexes of ligands bound to the enzyme. The study revealed that residual tags used for protein expression can lodge in the active site and hinder ligand binding. Additionally, the space groups in which the proteins are crystallized can significantly impact the visualization of the organophosphate ligands bound to the phosphotriesterase. The study also reveals that the crystallization precipitants can compete with and even preclude ligand binding, leading to false positives or the incorrect identification of lead drug candidates, which is particularly crucial for ligands with pharmacological and toxicological contexts. Overall, this study provides valuable insights into the challenges and considerations involved in studying the 3D structure of proteins with bound ligands, highlighting the importance of careful experimental design and rigorous data analysis to ensure the accuracy and reliability of the resulting protein-ligand structures.

biochemistry↗

bioRxiv2022

Do Newly Born Orphan Proteins Resemble Never Born Proteins?A Study using Deep Learning Algorithms

Newly Born proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by expression of novel Open Reading Frames, and appear throughout evolution. We were curious if the three recently developed programs for predicting protein structures, viz., AlphaFold2, RoseTTAFold, and ESMFold, might be of value for comparison of such Newly Born proteins to random polypeptides with amino acid content similar to that of native proteins, which have been called Never Born proteins. The programs were used to compare the structures of two sets of Never Born proteins that had been expressed - Group 1, which had been shown experimentally to possess substantial secondary structure, and Group 3, which had been shown to be intrinsically disordered. Overall, the models generated were scored as being of low quality but revealed some general principles. Specifically, all four members of Group 1 were predicted to be compact by all three algorithms. The members of Group 3 were predicted to be very extended, as would be expected for intrinsically disordered proteins. The three programs were then used to predict the structures of three orphan proteins whose crystal structures had been solved, two of which display novel folds. Finally, they were used to predict the structures of seven orphan proteins with well-identified biological functions, whose 3D structures are not known. Two proteins, which were predicted to be disordered based on their sequences, are predicted by all three structure algorithms to be extended structures. The other five were predicted to be compact structures with two exceptions in the case of AlphaFold2. All three prediction algorithms make remarkably similar and high-quality predictions for one large protein, HCO_11565, from a nematode. It is conjectured that this is due to many homologs in the taxonomically restricted family of which it is a member and to the fact that the Dali server revealed several non-related proteins with similar folds. Overall, orphan and taxonomically restricted proteins are often predicted to have compact 3D structures, sometimes with a novel fold that is a consequence of their novel sequences, which are associated with the appearance of new biological functions.

molecular biology↗

bioRxiv2019

Molecular Dynamics Simulations of the Interaction of Mouse and Torpedo Acetylcholinesterase with Covalent Inhibitors Explain Their Differential Reactivity: Implications for Drug Design

Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced breathing motions of the mouse enzyme relative to the Torpedo enzyme might explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approached the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride was able to approach the active-site serine of mouse acetylcholinesterase - but remained trapped above the bottleneck in the case of the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. Author summaryEnzymes are protein molecules that catalyze chemical reactions in living organisms, and are essential for their physiological functions. Proteins have well defined three-dimensional structures, but display flexibility; it is believed that this flexibility, known as their dynamics, plays a role in their function. Here we studied the neuronal enzyme acetylcholinesterase, which breaks down the neurotransmitter, acetylcholine. The active site of this enzyme is deeply buried, and accessed by a narrow gorge. A particular inhibitor, phenylmethylsulfonyl fluoride, is known to inhibit mouse acetylcholinesterase, but not that of the electric fish, Torpedo, even though their structures are very similar. A theoretical technique called molecular dynamics (MD) shows that the mouse enzyme is more flexible than the Torpedo enzyme. Furthermore, when the movement of the inhibitor down the gorge towards the active site is simulated using MD, the phenylmethylsulfonyl fluoride can reach the active site in the mouse enzyme, but not in the Torpedo enzyme, in which it remains trapped midway down the gorge. Our study emphasizes the importance of taking into account not only structure, but also dynamics, in designing drugs targeted towards proteins.

biophysics↗

bioRxiv2018

The physiological butyrylcholinesterase tetramer is a dimer of dimers stabilized by a superhelical assembly

The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localisation and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryo-electron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 [A]. Our structure reveals that the BChE tetramer is organised as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central anti-parallel polyproline II helix (PRAD). The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the HuBChE tetramer. Our cryo-EM structure reveals the basis for assembly of the physiological tetramers, and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases, and may provide a promising template for designing other proteins with improved circulatory residence times.

biophysics↗