Biology subjects

Cronin, N. B.

Publications and source records attributed to Cronin, N. B..

5 recordsLinked to original sources

bioRxiv2026

HTLV-1 intasome recruits the PP2A-B56 holoenzyme and restricts its phosphatase activity

Integrase catalyses the insertion of a DNA copy of the retroviral genome into host cell chromatin. Human T-cell lymphotropic virus type 1 (HTLV-1) and other deltaretroviral integrases associate with protein phosphatase 2A holoenzymes containing B56 regulatory subunits (PP2A-B56). Here, we show that integrase mutants defective in binding to most B56 isoforms retain intrinsic DNA strand transfer activity but are impaired in establishing infection. Using single-particle cryo-EM, we determined the structure of the simian T-cell lymphotropic virus type 1 (STLV-1) intasome in a 0.5-MDa complex with two copies of the heterotrimeric PP2A-B56{gamma} holoenzyme at 2.8 [A] resolution. The structure reveals that, in addition to engaging B56, integrase forms direct contacts with the catalytic subunit of PP2A and sterically occludes the phosphatase active site, preventing substrate access. Consistent with these findings, we show that the HTLV-1 intasome suppresses PP2A catalytic activity in a manner dependent on the integrase LxxIxE short linear motif. We further demonstrate that pharmacological inhibition of PP2A does not impair HTLV-1 infection. Together, our results indicate that recruitment of PP2A-B56 by the intasome serves a structural rather than catalytic function.

microbiology↗

bioRxiv2026

Cryo-EM structures of the CDK11-cyclin L-SAP30BP complex reveal mechanisms of CDK11 regulation

The cyclin-dependent kinase CDK11 functions in transcription, mitotic progression, and mRNA splicing. Specifically, spliceosome activation during the B to Bact transition depends on phosphorylation of the U2 snRNP component SF3B1 by the CDK11-cyclin L-SAP30BP complex. Here, we present the structure of this spliceosome-activating CDK-cyclin complex, determined by cryogenic electron microscopy at 2.3 [A] resolution. Our structure and biochemical experiments show that SAP30BP forms extensive interactions with cyclin L2, thereby stabilising it, and forms critical interactions with the C-terminal kinase lobe of CDK11 that promote complex assembly. Destabilisation of cyclin L2 in the absence of SAP30BP suggests that these principles are applicable to all CDK11-cyclin L complexes. Furthermore, we identify a pseudo-substrate sequence near the CDK11 C-terminus and provide evidence for a role of this segment in CDK11 auto-regulation. Finally, the structure of the CDK11-cyclin L2-SAP30BP complex bound to the clinical high-affinity CDK11 inhibitor OTS964 and a comparison to OTS964-bound off-target complexes provide insight into the mechanism of OTS964 selectivity and specificity.

molecular biology↗

bioRxiv2021

Cryo-EM structure of the Smc5/6 holo-complex

The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral integration. Here, we report the cryo-EM structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing a full overview of the complex in its apo / non-liganded form, and revealing how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the W-loop of Smc4 or F-loop of Smc1, mediates an essential interaction with Nse1. Taken together, these data confirm a degree of functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

biochemistry↗

bioRxiv2021

Positive and negative control of helicase recruitment at a bacterial chromosome origin

The mechanisms responsible for helicase loading during the initiation of chromosome replication in bacteria are unclear. Here we report both a positive and a negative mechanism for directing helicase recruitment in the model organism Bacillus subtilis. Systematic mutagenesis of the essential replication initiation gene dnaD and characterization of DnaD variants revealed protein interfaces required for interacting with the master initiator DnaA and with a specific single-stranded DNA (ssDNA) sequence located in the chromosome origin (DnaD Recognition Element, "DRE"). We propose that the location of the DRE within the replication origin orchestrates recruitment of helicase to achieve bidirectional DNA replication. We also report that the developmentally expressed repressor of DNA replication initiation, SirA, acts by blocking the interaction of DnaD with DnaA, thereby inhibiting helicase recruitment to the origin. These findings significantly advance our mechanistic understanding of helicase recruitment and regulation during bacterial DNA replication initiation. Because DnaD is essential for the viability of clinically relevant Gram-positive pathogens, DnaD is an attractive target for drug development.

microbiology↗

bioRxiv2018

Structural basis of Cullin-2 RING E3 ligase regulation by the COP9 signalosome

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 (N8) from activated Cullins. The structure of stable CSN-CRL can be used to understand this mechanism of regulation. Here we present the first structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (MS). These structures reveal a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen deuterium exchange-MS we show that CRL2 binding and conformational activation of CSN5/CSN6 occur in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identified novel sensory regions of the CSN that mediate its stepwise activation mechanism and provide a framework for better understanding the regulatory mechanism of other Cullin family members.\n\nOne sentence summaryStructure and dynamics of the CSN-CRL2 complexes assessed by cryo-electron microscopy and structural mass spectrometry.

biochemistry↗