Biology subjects

Farrell, D. P.

Publications and source records attributed to Farrell, D. P..

5 recordsLinked to original sources

bioRxiv2024

Automated identification of small molecules in cryo-electron microscopy data with density- and energy-guided evaluation

Methodological improvements in cryo-electron microscopy (cryoEM) have made it a useful tool in ligand-bound structure determination for biology and drug design. However, determining the conformation and identity of bound ligands is still challenging at the resolutions typical for cry-oEM. Automated methods can aid in ligand conformational modeling, but current ligand identification tools -- developed for X-ray crystallography data -- perform poorly at resolutions common for cryoEM. Here, we present EMERALD-ID, a method capable of docking and evaluating small molecule conformations for ligand identification. EMERALD-ID identifies 43% of common ligands exactly and identifies closely related ligands in 66% of cases. We then use this tool to discover possible ligand identification errors, as well as previously unidentified ligands. Furthermore, we show EMERALD-ID is capable of identifying ligands from custom ligand libraries of various small molecule types, including human metabolites and drug fragments. Our method provides a valuable addition to cryoEM modeling tools to improve small molecule model accuracy and quality.

biochemistry↗

bioRxiv2021

Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

biochemistry↗

bioRxiv2020

Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex

Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7-GTP. Here we report the 2.8 [A] crystal structure of the Rubicon RH domain in complex with Rab7-GTP. Our structure reveals a novel fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab-effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7 binding site restore efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

cell biology↗

bioRxiv2019

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation.

The bacterial flagellum is a remarkable molecular motor, present at the surface of many bacteria, whose primary function is to allow motility through the rotation of a long filament protruding from the bacterial cell. A cap complex, consisting of an oligomeric assembly of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex. This structure reveals that FliD is pentameric, with the N-terminal region of the protomer forming an unexpected extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded and propose a molecular model for the filament-cap interaction.

microbiology↗

bioRxiv2019

Independent evolution of polymerization in the Actin ATPase clan regulates hexokinase activity

The actin protein fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, like the carbohydrate kinases, do not polymerize. We find that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with unique ultrastructure, distinct from that of cytoskeletal polymers. In cells, Glk1 polymerizes upon sugar addition and depolymerizes upon sugar withdrawal. Glk1 polymerization inhibits its enzymatic activity, thus the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation eliminating Glk1 polymerization alleviates concentration-dependent enzyme inhibition, causing glucokinase activity to become unconstrained. Polymerization-based regulation of Glk1 activity serves an important function in vivo: yeast containing non-polymerizing Glk1 are less fit when growing on sugars and more likely to die when refed glucose. Glucokinase polymerization arose within the ascomycete fungi and is conserved across a group of divergent (150-200 mya) yeast. We show that Glk1 polymerization arose independently from other actin-related filaments and allows yeast to rapidly modulate glucokinase activity as nutrient availability changes.\n\nOne-sentence summaryYeast glucokinase activity is limited by its polymerization, which is critical for cell viability during glucose refeeding.

biochemistry↗