Biology subjects

Durie, C. L.

Publications and source records attributed to Durie, C. L..

5 recordsLinked to original sources

bioRxiv2025

Substrate-enhanced filamentation of 3-methylcrotonyl-CoA carboxylase in Legionella pneumophila

3-Methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent carboxylase that metabolizes the amino acid leucine. MCC is present in bacteria, fungi, plants, and animals. In humans, its overexpression is linked to cancer, and its deficiency is linked to inborn errors of metabolism with severe consequences, so understanding its structure and function has far reaching implications. Here, we explore the MCC from Legionella pneumophila, a pathogenic bacterium with a biphasic life cycle. Our endogenous holoenzyme yielded the highest resolution cryo-EM structure of MCC to date, allowing for identification of protein components by the machine learning tool ModelAngelo, confirmed independently by mass spectrometry. We also observed, for the first time, enhanced filamentation of MCC upon substrate binding. We propose that this filamentation, previously observed in the eukaryotes, but not in bacteria, may be important for cellular processes such as differentiation of life cycle or cell division.

biochemistry↗

bioRxiv2023

A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase

The mechanism by which polymerase -primase (pol-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of pol-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5'-end facilitates handoff of the primer to pol and increases pol processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of pol and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by pol-primase.

biochemistry↗

bioRxiv2021

Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 [A] resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure. We hypothesize this conformational flexibility is likely to facilitate the Dot/Icm T4SSs ability to translocate a remarkably large set of [~]300 putative substrates across the inner and outer membranes of the bacterial cell.

microbiology↗

bioRxiv2020

Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex

The pathogenesis of Helicobacter pylori-associated gastric cancer is dependent on delivery of CagA into host cells through a type IV secretion system (T4SS). The H. pylori Cag T4SS includes a large membrane-spanning core complex containing 5 proteins, organized into an outer membrane cap (OMC), a periplasmic ring (PR) and a stalk. Here, we report cryo-EM reconstructions of a core complex lacking Cag3 and an improved map of the wild-type complex. We define the structures of two unique species-specific components (Cag3 and CagM) and show that Cag3 is structurally similar to CagT. Unexpectedly, components of the OMC are organized in a 1:1:2:2:5 molar ratio (CagY:CagX:CagT:CagM:Cag3). CagX and CagY are components of both the OMC and the PR and bridge the symmetry mismatch between these regions. These results reveal that assembly of the H. pylori T4SS core complex is dependent on incorporation of interwoven species-specific components.

microbiology↗

bioRxiv2020

Structural analysis of the Legionella pneumophila Dot/Icm Type IV Secretion System Core Complex

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia Legionnaires Disease. This infection and subsequent pathology require the Dot/Icm Type IV Secretion System (T4SS) to deliver effector proteins into host cells. Compared to prototypical T4SSs, the Dot/Icm assembly is much larger, containing ~27 different components including a core complex reported to be composed of five proteins: DotC, DotD, DotF, DotG, and DotH. Using single particle cryo-electron microscopy (cryo-EM), we report reconstructions of the core complex of the Dot/Icm T4SS that includes a symmetry mismatch between distinct structural features of the outer membrane cap (OMC) and periplasmic ring (PR). We present models of known core complex proteins, DotC, DotD, and DotH, and two structurally similar proteins within the core complex, DotK and Lpg0657. This analysis reveals the stoichiometry and contact interfaces between the key proteins of the Dot/Icm T4SS core complex and provides a framework for understanding a complex molecular machine.

microbiology↗