Biology subjects

Rathinaswamy, M. K.

Publications and source records attributed to Rathinaswamy, M. K..

7 recordsLinked to original sources

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

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

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↗

bioRxiv2021

Accurate prediction of protein structures and interactions using a 3-track network

DeepMind presented remarkably accurate protein structure predictions at the CASP14 conference. We explored network architectures incorporating related ideas and obtained the best performance with a 3-track network in which information at the 1D sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The 3-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables rapid solution of challenging X-ray crystallography and cryo-EM structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate models of protein-protein complexes from sequence information alone, short circuiting traditional approaches which require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research. One-Sentence SummaryAccurate protein structure modeling enables rapid solution of structure determination problems and provides insights into biological function.

bioinformatics↗

bioRxiv2021

Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

The class IB phosphoinositide 3-kinase (PI3K), PI3K{gamma}, is a master regulator of immune cell function, and a promising drug target for both cancer and inflammatory diseases. Critical to PI3K{gamma} function is the association of the p110{gamma} catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G-protein coupled receptors (GPCRs). Here, we report the cryo-EM structure of a heterodimeric PI3K{gamma} complex, p110{gamma}-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its G{beta}{gamma} binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary G{beta}{gamma} binding site in p110{gamma}. Multiple oncogenic mutations mapped to these novel interfaces and enhanced G{beta}{gamma} activation. A nanobody that specifically binds to the p101-G{beta}{gamma} interface blocks activation providing a novel tool to study and target p110{gamma}-p101-specific signaling events in vivo.

biochemistry↗

bioRxiv2021

HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism and immunity. The class IB PI3K, PI3K{gamma}, is a heterodimeric complex composed of a catalytic p110{gamma} subunit bound to a p101 or p84 regulatory subunit. PI3K{gamma} is a critical component in multiple immune signaling processes and is dependent on activation by Ras and GPCRs to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3K{gamma} single chain camelid nanobodies using hydrogen deuterium exchange mass spectrometry (HDX-MS) for structural and biochemical studies. This allowed us to identify nanobodies that stimulated lipid kinase activity, blocked Ras activation and specifically inhibited p101-mediated GPCR activation. Overall, this reveals novel insight into PI3K{gamma} regulation and identifies sites that may be exploited for therapeutic development. Highlights- HDX-MS rapidly identifies epitopes of camelid single-chain nanobodies raised against Class IB PI3K complexes, p110{gamma}/p101 and p110{gamma}/p84 - A nanobody targeting p101 improves local resolution in EM studies with p110{gamma}/p101 facilitating structural characterization of the complex - Nanobodies that bind at the interfaces with the lipidated activators Ras and G{beta}{gamma} can prevent activation of p110{gamma}/p101 and p110{gamma}/p84

biochemistry↗

bioRxiv2020

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to selective inhibitors

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the p110{gamma} subunit playing a key role in immune signalling. PI3K{gamma} is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the c-terminus can inactivate or activate enzyme activity. Screening of small molecule inhibitors using HDX-MS revealed that activation loop binding inhibitors induce allosteric conformational changes that mimic those seen for the R1021C mutant. Structural analysis of clinically advanced PI3K inhibitors revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insight into the regulatory mechanisms that control PI3K{gamma} kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

biochemistry↗