Biology subjects

Jenkins, M. L.

Publications and source records attributed to Jenkins, M. L..

14 recordsLinked to original sources

bioRxiv2026

Phosphorylation of the C-terminus of PI4KA inhibits lipid kinase activity

Phosphatidylinositol 4-kinase alpha (PI4KA) is a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI) at the plasma membrane (PM). PI4P generated by PI4KA is essential for both plasma membrane identity and for PIP2 and PIP3 signalling driven by the PLC and PI3K family of enzymes. While the structure of PI4KA is known, the regulatory mechanisms that control its activity are undefined. Here, we discovered PI4KA can be inhibited through phosphorylation of Y2090 in the kinase domain, which can be phosphorylated by multiple tyrosine kinases. Y2090 phosphorylation causes local conformational changes in the k12 C-terminal helix. PI4KA activity is predominantly controlled through membrane recruitment by EFR3, with Y2090 phosphorylation inhibiting EFR3 bound PI4KA. Phosphorylation of the k12 C-terminal helix is found in PI3Ks and PI4Ks, suggesting an evolutionarily conserved regulatory mechanism for diverse phosphoinositide kinases. Overall, our work reveals novel molecular insight into inhibitory post-translational regulation of PI4KA.

biochemistry↗

bioRxiv2026

Mapping SARS-CoV-2 Nucleocapsid Function with Nanobodies

The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the proteins structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential. ImportanceSARS-CoV-2 and emerging coronaviruses remain a major global health threat, yet critical gaps persist in our understanding of their molecular pathogenesis. The nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 antigen, plays essential roles beyond genome packaging, including immune evasion and intracellular organization. Here, we generate and characterize a panel of domain-specific nanobodies (VHHs) that enable precise dissection of Ns functional architecture. Using integrated biochemical, structural, and virological approaches, we uncover distinct mechanisms of viral inhibition, including disruption of phase condensation through a conserved linker region. These findings address long-standing knowledge gaps about a multifunctional viral protein and establish VHHs as powerful, modular tools for probing coronavirus biology, with broad potential for therapeutic, diagnostic, and cell biology applications.

microbiology↗

bioRxiv2024

Molecular basis for plasma membrane recruitment of PI4KA by EFR3

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIII/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease. One sentence summaryThe cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA complex reveals the molecular mechanism underlying PI4KA membrane localisation, and provides novel insight into PI4KA regulation and its involvement in human disease.

biochemistry↗

bioRxiv2024

Structure of Calcineurin bound to PI4KA reveals dual interface in both PI4KA and FAM126A

Phosphatidylinositol 4-kinase alpha (PI4KA) maintains the PI4P and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into a complex with TTC7 and FAM126 proteins. This complex can be regulated by the CNA{beta}1 isoform of the phosphatase Calcineurin. We previously identified that CNA{beta}1 directly binds to FAM126A. Here, we report a cryo-EM structure of a truncated PI4KA complex bound to Calcineurin, revealing a direct Calcineurin interaction with PI4KA. Additional HDX-MS and computational analysis show that Calcineurin forms a complex with an evolutionarily conserved IKISVT sequence in PI4KAs horn domain. We also characterised conserved LTLT and PSISIT Calcineurin binding sequences in the C-terminus of FAM126A. These sites are in close proximity to phosphorylation sites in the PI4KA complex, suggesting key roles of Calcineurin-regulated phosphosites in PI4KA regulation. This work reveals novel insight into how Calcineurin can regulate PI4KA activity.

biochemistry↗

bioRxiv2023

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3K{gamma} is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKC{beta} activated by the IgE receptor, and G{beta}{gamma} subunits released from activated GPCRs. PI3K{gamma} can form two distinct complexes, with the p110{gamma} catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110{gamma} in regulating lipid kinase activity of distinct PI3K{gamma} complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110{gamma} membrane recruitment or Ras/G{beta}{gamma} binding, but instead decreased ATP turnover. We also identified that p110{gamma} can be activated by dual PKC{beta} helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKC{beta} phosphorylation is selective for p110{gamma}-p84 compared to p110{gamma}-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKC{beta} mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110{gamma} that is distinct between p110{gamma}-p84 and p110{gamma}-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

biochemistry↗

bioRxiv2022

Structure and dynamics of human perilipin 3 membrane association

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyzed how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We found the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG enriched membranes. Membrane binding induces a disorder/order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.

biochemistry↗

bioRxiv2022

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs

Class IB phosphoinositide 3-kinase (PI3K{gamma}) is activated in immune cells by diverse stimuli and can form two distinct complexes, with the p110{gamma} catalytic subunit binding to either p101 or p84 regulatory subunits. These two complexes are differentially activated by G-protein coupled receptors (GPCRs) and Ras, but the molecular details of this activation are still unclear. Using a combination of X-ray crystallography, HDX-MS, EM, molecular modeling, and biochemical assays we reveal molecular differences between the two p110{gamma}-p84 and p110{gamma}-p101 complexes that explain their differential activation. The structure of p110{gamma}-p84 shows a similar assembly to p110{gamma}-p101 at the p110{gamma} interface, however the interface in p110{gamma}-p84 is dynamic and is evolutionarily conserved to be less stable compared to p110{gamma}-p101. The p110{gamma}-p84 complex is only weakly recruited to membranes by G{beta}{gamma} subunits alone and requires recruitment by Ras to allow for G{beta}{gamma} activation through an interaction with the p110{gamma} helical domain. The interfaces of the p101 GBD with G{beta}{gamma}, and the p110{gamma} helical domain with G{beta}{gamma} were determined using computational alphafold2 modeling and HDX-MS. There are distinct differences in the C-terminal domain of p84 and p101, which allows p101 to bind G{beta}{gamma} subunits, while p84 does not. The two G{beta}{gamma} interfaces in p110{gamma} and p101 are distinct, revealing how unique mutants of G{beta}{gamma} cause differential disruption of PI3K{gamma} complex activation. Overall, our work provides key insight into the molecular basis for how different PI3K{gamma} complexes are activated.

biochemistry↗

bioRxiv2022

ATP-competitive and allosteric inhibitors induce differential conformational changes at the auto-inhibitory interface of Akt

The protein kinase Akt is a master regulator of pro-growth signalling in the cell. Akt is activated through its targeted recruitment to phosphoinositides, leading to disruption of the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains. Hyper activation of Akt is common in oncogenic transformation, with multiple oncogenic activating mutants identified in Akt. This has led to the development of potent and selective ATP-competitive and allosteric inhibitors for Akt. Paradoxically, some ATP-competitive Akt inhibitors cause hyperphosphorylation of Akt. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding to the Akt active site inhibitor A-443654, and the Akt allosteric inhibitor MK-2206. We compared the conformational changes that occurred for each inhibitor under three different states of Akt: i-inactive monophosphorylated, ii-partially active tris-phosphorylated [T308, T450, S473], and iii-fully activated, tris-phosphorylated bound to PIP3 membranes. The allosteric MK-2206 inhibitor results in large scale allosteric conformational changes in all states, and restricts membrane binding through sequestration of the PH domain. Binding of the A-443654 inhibitor led to large scale allosteric conformational changes in both the monophosphorylated and phosphorylated states, leading to an alteration in the autoinhibitory PH-kinase interface. We also observed increased protection in the PH domain upon membrane binding in the presence of A-443654, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and dynamic conformational changes induced by Akt inhibitors, and has important implications for the design of Akt targeted therapeutics.

biochemistry↗

bioRxiv2022

Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus

PIK3CA encoding the phosphoinositide 3-kinase (PI3K) p110 catalytic subunit is frequently mutated in cancer, with mutations occurring widely throughout the primary sequence. The full set of mechanisms underlying how PI3Ks are activated by all oncogenic mutations on membranes are unclear. Using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS), we reveal unique regulatory mechanisms underlying PI3K activation. Engagement of p110 on membranes leads to disengagement of the ABD of p110 from the catalytic core, and the C2 domain from the iSH2 domain of the p85 regulatory subunit. PI3K activation also requires reorientation of the p110 C-terminus, with mutations that alter the inhibited conformation of the C-terminus increasing membrane binding. Mutations at the C-terminus (M1043I/L, H1047R, G1049R, and N1068KLKR) activate p110 through distinct mechanisms, with this having important implications for mutant selective inhibitor development. This work reveals unique mechanisms underlying how PI3K is activated by oncogenic mutations, and explains how double mutants can synergistically increase PI3K activity.

biochemistry↗

bioRxiv2022

Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes

Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-gamma (PLC-{gamma}) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that PLC-{gamma}1 is relatively inert to lipid vesicles that contain its substrate, PIP2, unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-{gamma}1 and is exaggerated in PLC-{gamma}1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-{gamma}1 to favor activation. This receptor-induced priming of PLC-{gamma}1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-{gamma}1 operating on lipid vesicles but not a soluble analog of PIP2 and highlights cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-{gamma} isozymes.

biochemistry↗

bioRxiv2021

Palmitoylation targets the Calcineurin phosphatase to the Phosphatidylinositol 4-kinase complex at the plasma membrane

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNA{beta}1. We show that unlike canonical cytosolic calcineurin, CNA{beta}1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNA{beta}1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNA{beta}1.

biochemistry↗

bioRxiv2021

Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that the TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

biochemistry↗

bioRxiv2019

Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition

Phospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLC{beta} by Gq and/or G{beta}{gamma} subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLC{beta} isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLC{beta}3 bound to Gq is known, however, for both Gq and G{beta}{gamma}, the mechanism for PLC{beta} activation on membranes is unknown. We examined PLC{beta}2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). G{beta}{gamma} caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gq showed decreased deuterium incorporation at the Gq binding site on PLC{beta}. In vitro G{beta}{gamma}-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLC{beta} hydrolase activity.

biochemistry↗

bioRxiv2019

Characterisation of the c10orf76-PI4KB complex, and its necessity for Golgi PI4P levels and enterovirus replication

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi, and is essential for the viral replication of specific enteroviruses. We used hydrogen deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 was also required for proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.\n\nHighlightsO_LIc10orf76 forms a direct complex with PI4KB, with the interface formed by a disorder-to-order transition in the kinase linker of PI4KB\nC_LIO_LIThe c10orf76 binding site of PI4KB can be phosphorylated by PKA, with phosphorylation leading to decreased affinity for c10orf76\nC_LIO_LIComplex-disrupting mutants of PI4KB and c10orf76 reveal that PI4KB recruits c10orf76 to the Golgi/TGN\nC_LIO_LIDepletion of c10orf76 leads to decreases in both active Arf1 and Golgi PI4P levels\nC_LIO_LIEnteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76-PI4KB complex\nC_LI

biochemistry↗