Biology subjects

Endicott, J. A.

Publications and source records attributed to Endicott, J. A..

5 recordsLinked to original sources

bioRxiv2026

FragLite mapping to identify the BRD4 recruitment site of P-TEFb

The eukaryotic positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T, plays a central role in regulating RNA polymerase II (RNAPII). Phosphorylation of the RNAPII C-terminal domain (CTD) by P-TEFb promotes promoter proximal pause release and enables productive transcriptional elongation across many genes. Cyclin T mediates protein-protein interactions, several of which have been structurally characterised, that help to recruit and fine-tune P-TEFb activity to ensure a tight regulation of transcription. We have previously reported a set of halogenated chemical fragments termed FragLites that can prospectively identify protein interaction sites. Here, we report the FragLite map of cyclin T2, revealing binding sites corresponding to structurally defined cyclin T partners CDK9, AFF4, and HIV-1 Tat. Furthermore, we demonstrate the utility of FragLites in identifying a previously uncharacterised BRD4 binding site. By integrating FragLite clustering with biophysical analyses and AlphaFold3 modelling, we delineate the cyclin T-BRD4 interface. These analyses provide a comprehensive, chemically enriched fragment map highlighting functionally relevant sites to support future probe and modulator development to selectively target P-TEFb.

biophysics↗

bioRxiv2024

Crystallographic fragment screening of CDK2-cyclin A: FragLites map sites of protein-protein interaction

Protein-protein interaction sites (PPIs) are potentially more selective therapeutic binding sites than protein substrate binding sites. PPIs include distinct regions frequently called "hotspots," sites of key amino acid interactions. Prospective identification of these hotspots through X-ray crystallographic screening could assist in the identification of separation of function mutants for experimental validation, enhance confidence in AI-generated multiprotein complex predictions and accelerate development of selective chemical probes. To explore these applications, we utilize the FragLite library to examine the binding surfaces of CDK2-cyclin A. The many protein- and peptide-CDK2-cyclin A complexes that have been structurally characterised make this complex an appropriate test case. We show that FragLites comprehensively map both known sites of protein-protein interaction on CDK2-cyclin A and identify a possible uncharacterised site, providing a structural method toward directing mechanistic studies and providing starting points for chemical probe design.

biophysics↗

bioRxiv2023

Cryo-EM structure of the CDK2-cyclin A-CDC25A complex

The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich motif of cyclin-dependent protein kinases (CDKs) to activate CDK activity. Here, we present the cryogenic-electron microscopy (cryo-EM) structure of CDK2-cyclin A in complex with CDC25A at 2.91 [A] resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further reveal an unanticipated CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25- targeting anticancer strategies.

biochemistry↗

bioRxiv2021

Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a

We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 plays a critical role in the cell cycle by inhibiting cyclin-dependent kinases CDK4 and CDK6, and it is inactivated in sporadic and familial cancers. The majority of the p16 missense mutations cause loss of function by destabilizing the proteins structure. We show that the nanobodies bind p16 with nanomolar affinities and restore the stability of a range of different cancer-associated p16 mutations located at sites throughout the protein. The nanobodies also bind and stabilize p16 in a cellular setting. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. These findings indicate that nanobodies could be used as pharmacological chaperones to determine the consequences of restoring the function of p16 in the cell. HighlightsO_LIDescribes a nanobody (single-domain antibody) capable of binding tumor suppressor protein p16 C_LIO_LINanobody binding can stabilise wild-type and cancer-associated p16 mutants C_LIO_LINanobody-p16 crystal structure reveals why nanobody binding is compatible with p16-CDK6 interaction C_LI

biochemistry↗

bioRxiv2020

Discriminative SKP2 interactions with CDK-cyclin complexes support a cyclin A-specific role in p27KIP1 degradation

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

biochemistry↗