Biology subjects

Grim, J.

Publications and source records attributed to Grim, J..

3 recordsLinked to original sources

bioRxiv2025

Quantitative Membrane Binding Assays Reveal an Inhibitory Role for the BRAF-Specific Region in CRD and Lipid Interaction

BRAF is a serine/threonine kinase and a central effector of the mitogen-activated protein kinase (MAPK) signaling pathway, frequently mutated in cancer. Its activation is tightly controlled by autoinhibitory mechanisms that regulate membrane recruitment and dimerization. The BRAF-specific region (BSR), located at the N-terminus, is known to promote isoform-preferred RAS binding and facilitate dimerization with kinase suppressor of RAS (KSR), yet its role in regulating lipid interaction has remained unexplored. Here, we identify the BSR as a previously unrecognized inhibitory module that attenuates lipid binding by the cysteine-rich domain (CRD). Using quantitative in vitro reconstitution with supported lipid bilayers and fluorescence microscopy, we demonstrate that the BRAF CRD exhibits high intrinsic affinity for phosphatidylserine-rich membranes, but the inclusion of the BSR markedly reduces the membrane binding. We further demonstrate that the inhibitory function of the BSR correlates with its global electrostatic properties rather than a single defined sequence motif. This inhibitory effect of BSR was corroborated in live cells by quantifying plasma membrane localization of BRAF constructs, including the full-length protein. When canonical autoinhibition of CRD--mediated by sequestration within the 14-3-3 dimer--is disrupted by oncogenic mutation or RAF inhibitor treatment, the BSR assumes a compensatory role in repressing CRD-lipid interaction. This additional regulatory layer provided by the BSR prevents RAS-independent membrane recruitment under both physiological and pathological conditions. Broad Impact StatementProtein-lipid interactions are a fundamental mechanism for regulating the localization and activity of signaling proteins. This study reveals that the N-terminal segment of BRAF acts as an inhibitory module that suppresses lipid engagement by CRD, particularly when canonical autoinhibition is disrupted by oncogenic mutation or inhibitor treatment. This additional layer of regulation provides new insight into BRAF membrane dynamics and may have implications in therapeutic intervention of dysregulated BRAF signaling.

biochemistry↗

bioRxiv2025

Positive cooperativity between RAS-binding and cysteine-rich domains regulates RAF membrane binding kinetics via lateral rebinding

RAF activation requires interactions with both RAS nanoclusters and membrane lipids, yet the molecular basis of this process remains unclear. Using a bottom-up reconstitution approach, we show how coordinated protein-protein and protein-lipid interactions regulate membrane binding dynamics of RAF to drive its multistep activation. Within membrane environments, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) exhibit cooperativity, with CRD-mediated phosphatidylserine binding stabilizing the RBD:RAS complex. Importantly, RAF remains membrane-bound through lateral rebinding to RAS, where a weak CRD-lipid interaction plays an essential role. This lateral rebinding extends RAFs membrane dwell time under high RAS density conditions, which are found in RAS nanoclusters. This prolonged membrane residence likely facilitates kinetic proofreading of RAFs multistep activation within RAS nanoclusters, ensuring signaling specificity. Given the high abundance of weak multivalent membrane interactions, lateral rebinding may be a common mechanism for regulating the activity of signaling proteins through sustained membrane retention.

biochemistry↗

bioRxiv2022

Unraveling the Palindromic and Non-Palindromic Motifsof Retroviral Integration Site Sequences by Statistical Mixture Models

A weak palindromic nucleotide motif is the hallmark of retroviral integration site alignments. Previously, the motifs were explained by an overlap of the non-palindromic motif being present on one of the half-site of targeted sequences. Here, we applied multicomponent mixture models to integration site sequences of diverse retroviruses. We demonstrate that the weak palindromic motifs result from a combination of independent sub-motifs restricted to only a few positions proximal to the site of integration. The sub-motifs are formed by either palindrome-forming nucleotide preference or nucleotide exclusion. Using the mixture models, we also identified HIV-1-favored palindromic sequences in Alu repeats serving as hotspots for integration. Our work presents a novel statistical approach to the analysis of retroviral integration site sequences, which can form a valuable tool in the analysis of DNA motifs. The presented results shed new light on the selection of target site sequences for retroviral integration.

microbiology↗