HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases
TL;DR: In this paper, the authors 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.
Abstract: 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
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TL;DR: Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, the authors identify molecular differences between PI3Kγ-p84 and p110γp101 that explain their differential membrane recruitment and activation by Ras and GPCRs.
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TL;DR: In this article , the authors summarize recent advances in chemical probe development for emerging target classes such as solute carriers and ubiquitin-related targets and highlight open resources to inform and facilitate chemical probe discovery.
Abstract: ABSTRACT Introduction The rational development of new therapeutics requires a thorough understanding of how aberrant signalling affects cellular homeostasis and causes human disease. Chemical probes are tool compounds with well-defined mechanism-of-action enabling modulation of, for example, domain-specific protein properties in a temporal manner, thereby complementing other target validation methods such as genetic gain- and loss-of-function approaches. Areas covered In this review, the authors summarize recent advances in chemical probe development for emerging target classes such as solute carriers and ubiquitin-related targets and highlight open resources to inform and facilitate chemical probe discovery as well as tool compound selection for target validation and phenotypic screening. Expert opinion Chemical probes are powerful tools for drug discovery that have led to fundamental insights into biological processes and have paved the way for the development of first-in-class drugs. Open resources can inform on various aspects of chemical probe development and provide access to data and recommendations on use of chemical probes to catalyse collaborative science and help accelerate drug target identification and validation.
TL;DR: In this paper , the authors 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.
Abstract: PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ 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 p110γ in regulating lipid kinase activity of distinct PI3Kγ 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 p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-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.
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TL;DR: Key statistics on the current data contents and volume of downloads are outlined, and how PRIDE data are starting to be disseminated to added-value resources including Ensembl, UniProt and Expression Atlas are outlined.
Abstract: The PRoteomics IDEntifications (PRIDE) database (https://www.ebi.ac.uk/pride/) is the world’s largest data repository of mass spectrometry-based proteomics data, and is one of the founding members of the global ProteomeXchange (PX) consortium. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2016. In the last 3 years, public data sharing through PRIDE (as part of PX) has definitely become the norm in the field. In parallel, data re-use of public proteomics data has increased enormously, with multiple applications. We first describe the new architecture of PRIDE Archive, the archival component of PRIDE. PRIDE Archive and the related data submission framework have been further developed to support the increase in submitted data volumes and additional data types. A new scalable and fault tolerant storage backend, Application Programming Interface and web interface have been implemented, as a part of an ongoing process. Additionally, we emphasize the improved support for quantitative proteomics data through the mzTab format. At last, we outline key statistics on the current data contents and volume of downloads, and how PRIDE data are starting to be disseminated to added-value resources including Ensembl, UniProt and Expression Atlas.
5,735 citations
TL;DR: The presence of considerable amounts of IgG-like material of Mr 100K in the serum of the camel, which is composed of heavy-chain dimers and devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, opens new perspectives in the engineering of antibodies.
Abstract: Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres. Except in the case of 'heavy chain' disease, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains or cloned VH domains. Here we investigate the presence of considerable amounts of IgG-like material of M(r) 100K in the serum of the camel (Camelus dromedarius). These molecules are composed of heavy-chain dimers and are devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, a finding that calls into question the role of light chains in the camel. Camel heavy-chain IgGs lack CH1, which in one IgG class might be structurally replaced by an extended hinge. Heavy-chain IgGs are a feature of all camelids. These findings open new perspectives in the engineering of antibodies.
2,863 citations
TL;DR: This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR and the most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain.
Abstract: G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The b2 adrenergic receptor (b2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomericb2AR and nucleotide-free Gs heterotrimer. The principal interactions between the b2AR and Gs involve the amino- and carboxy-terminal a-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The
2,676 citations
TL;DR: It is found that large-scale genomic analysis can identify nearly all known cancer genes in these cancer types and 33 genes that were not previously known to be significantly mutated in cancer, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis.
Abstract: Although a few cancer genes are mutated in a high proportion of tumours of a given type (.20%), most are mutated at intermediate frequencies (2–20%). To explore the feasibility of creating a comprehensive catalogue of cancer genes, we analysed somatic point mutations in exome sequences from 4,742 human cancers and their matched normal-tissue samples across 21 cancer types. We found that large-scale genomic analysis can identify nearly all known cancer genes in these tumour types. Our analysis also identified 33 genes that were not previously known to be significantly mutated in cancer, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis. Down-sampling analysis indicates that larger sample sizes will reveal many more genes mutated at clinically important frequencies. We estimate that near-saturation may be achieved with 600– 5,000 samples per tumour type, depending on background mutation frequency. The results may help to guide the next stage of cancer genomics. Comprehensive knowledge of the genes underlying human cancers is a critical foundation for cancer diagnostics, therapeutics, clinical-trial design and selection of rational combination therapies. It is now possible to use genomic analysis to identify cancer genes in an unbiased fashion, based on the presence of somatic mutations at a rate significantly higher than the expected background level. Systematic studies have revealed many new cancer genes, as well as new classes of cancer genes 1,2 . They have also made clear that, although some cancer genes are mutated at high frequencies, most cancer genes in most patients occur at intermediate frequencies (2–20%) or lower. Accordingly, a complete catalogue of mutations in this frequency class will be essential for recognizing dysregulated pathways and optimal targets for therapeutic intervention. However, recent work suggests major gaps in our knowledge of cancer genes of intermediate frequency. For example, a study of 183 lung adenocarcinomas 3 found that 15% of patients lacked even a single mutation affecting any of the 10 known hallmarks of cancer, and 38% had 3 or fewer such mutations. In this paper, we analysed somatic point mutations (substitutions and small insertion and deletions) in nearly 5,000 human cancers and their matched normal-tissue samples (‘tumour–normal pairs’) across 21 tumour types. The questions that we examine here are: first, whether large-scale genomic analysis across tumour types can reliably identify all known cancer genes; second, whether it will reveal many new candidate cancer genes; and third, how far we are from having a complete catalogue of cancer genes (at least those of intermediate frequency). We used rigorous statistical methods to enumerate candidate cancer genes and then carefully inspected each gene to identify those with strong biological connections to cancer and mutational patterns consistent with the expected function. The analysis reveals nearly all known cancer genes and revealed 33 novel candidates, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis. Importantly, the data show that the
2,565 citations
TL;DR: A camelid antibody fragment to the human β2 adrenergic receptor is generated, and an agonist-bound, active-state crystal structure of the receptor-nanobody complex is obtained, providing insights into the process of agonist binding and activation.
Abstract: G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human b2 adrenergic receptor (b2AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive b2AR structure reveals subtle changes in the binding
1,558 citations