Biology subjects

Hamilton, C. J.

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

3 recordsLinked to original sources

bioRxiv2025

IcmX Plug Ejection in L. pneumophila's Type IV Secretion System: Is It Possible?

1Legionella pneumophila is a gram-negative bacterial pathogen that is the causative agent of several infectious diseases, notably the severe form of pneumonia known as Legionnaires Disease. L. pneumophila targets amoebas and, in humans, alveolar macrophages. L. pneumophila infects its host by envelopment into the host cell through phagocytosis followed by the activation of the Type IV Secretion System (T4SS). The T4SS secretes effector proteins into the host cell which deactivate cell defenses and reprogram cell function to support L. pneumophila reproduction. After reproduction, L. pneumophila lyses the host cell and the cycle repeats, causing swelling and destroying a primary cell in the hosts immune system--infections caused by L. pneumophila are notoriously hard to treat. The stages of the infection process are known but the physical mechanisms are poorly understood. The structure of the T4SS includes a 13 member polymer, DotG which protrudes from the outer leaflet of the bacterial outer membrane. Given this exposure and the necessity of T4SS for infection, DotG is a promising drug target. However, designing an effective drug requires understanding the physical mechanisms. Via MD simulations we have tested hypotheses regarding its function. Specifically, we show the feasibility that an applied force on IcmX, a plug-like penta-mer that initially blocks transport through the T4SS channel, can cause a structural change in DotG and allow for protein release. Further, this applied force could reasonably come from a build up of pressure in the channel from other effector proteins. We conclude that the process of secretion is dependent on the induced structural change in DotG. Inhibiting this change provides a possible drug development direction.

biophysics↗

bioRxiv2025

MembraneBuilder: High-speed membrane construction for large molecular dynamics simulations

1Membrane building and embedding of proteins into a membrane is a crucial step in preparing a simulation for Molecular Dynamics (MD). When an MD simulation contains several million atoms, embedding the protein into a membrane becomes computationally expensive. MembraneBuilder [1] speeds up the generation of these membranes by simplifying the lipid insertion process, which allows membranes to be generated for these larger systems. Realistic lipid densities and elimination of infinite forces caused by overlapping atoms are achieved at a low computational cost. See Figure 1 for specific time comparisons. The comparisons emphasize that not only does MembraneBuilder run orders of magnitude faster than state-of-the-art programs [2, 3, 4], it enables generation of systems several orders of magnitude larger. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/689436v1_fig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1ebfd2aorg.highwire.dtl.DTLVardef@39e8faorg.highwire.dtl.DTLVardef@1ee8ccborg.highwire.dtl.DTLVardef@5d9294_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Speed Analysis: Comparison between the amount of time taken to run MembraneBuilder and CHARMM-GUI with varying protein sizes and their requisite amount of lipids. Note that the x and y scales are on a log scale, which further emphasizes the efficiency of MembraneBuilder. C_FIG MembraneBuilder enables customizable embedding of proteins into a membrane. Users can modify lipid types and their ratios, the location to insert the membrane, the size of the membrane box, as well as the lipid density. The program reduces the computational time required to perform a membrane embedding by bypassing many of the computationally expensive steps, such as identification of polar/non-polar sections of the protein and exhaustive packing of the lipids into the membrane with the intent to avoid steric clashes. Efficient packing begins with constricting the lipid bonds by a given factor in x, y, and z directions before inserting them on a grid. Grid spacing is determined by the width of the constricted lipids. The grid spacing is smaller than in other lipid-generation tools due to the constriction process. The constricted bonds will relax and the lipids will assume realistic interweaving during energy minimization and equilibration.

biophysics↗

bioRxiv2017

N-methyl-bacillithiol, a new metabolite discovered in the Chlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed.

Low-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae. Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed that Chlorobaculum tepidum contained a novel LMW thiol with a mass of 412 {+/-} 1 Da corresponding to a molecular formula of C14H24N2O10S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, the Cba. tepidum thiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in all Chlorobi examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes. A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobi, Bacteroidetes, Deinococcus-Thermus, and Firmicutes, but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria.\n\nSignificance StatementHere, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor in Cba. tepidum and the gene, nmbA, encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailed in vivo and in vitro dissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.

microbiology↗