Biology subjects

Kuriyan, J.

Publications and source records attributed to Kuriyan, J..

27 records · Page 2Linked to original sources

bioRxiv2020

A structural mechanism for the generation of biased agonism in the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor- (TGF-), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF- with similar affinity but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF- differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.

biochemistry↗

bioRxiv2020

GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation1,2. The central hub domain regulates the activity of CaMKII by organizing the holoenzyme complex into functional oligomers3-6. Recent findings have suggested that the hub is also an allosteric determinant of kinase activity7, and is thus an emerging target for therapies to correct CaMKII dysregulation8,9. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKII hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite {gamma}-hydroxybutyrate (GHB)10 we reveal that CaMKII is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 [A] crystal structure of CaMKII with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKII hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKII-selective ligands have therapeutic potential.

biochemistry↗

bioRxiv2020

Breakage of the Oligomeric CaMKII Hub by the Regulatory Segment of the Kinase

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a dodecameric or tetradecameric enzyme with crucial roles in neuronal signaling and cardiac function. Activation of CaMKII is reported to trigger the exchange of subunits between holoenzymes, which can increase spread of the active state. Using mass spectrometry, we now show that peptides derived from the sequence of the CaMKII- regulatory segment can bind to the CaMKII- hub assembly and break it into smaller oligomers. Molecular dynamics simulations show that the regulatory segments can dock spontaneously at the interface between hub subunits, trapping large fluctuations in hub structure. Single-molecule fluorescence intensity analysis of human CaMKII- isolated from mammalian cells shows that activation of CaMKII- results in the destabilization of the holoenzyme. Our results show how the release of the regulatory segment by activation and phosphorylation could allow it to destabilize the hub, producing smaller CaMKII assemblies that can reassemble to form new holoenzymes.

bioinformatics↗

bioRxiv2020

CRISPR-Cas12a exploits R-loop asymmetry to form double strand breaks

Most type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing applications. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one of the DNA strands of a double-helical substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we show that Cas12a-mediated R-loop formation destabilizes DNA at the second-strand cleavage site, which is located outside of the R-loop structure and beyond the 3' end of the guide RNA. Chemical and fluorescent DNA probes reveal that this destabilization is an intrinsic feature of DNA flanking the RNA-3' side of R-loops and does not require direct protein interactions. Interestingly, DNA flanking the RNA-5' side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

biochemistry↗

bioRxiv2019

Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation

The activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) depends on the balance between activating and inhibitory autophosphorylation (Thr 286 and Thr 305/306, respectively, in the human isoform). Variation in the lengths of the flexible linkers that connect the kinase domains of CaMKII to a central oligomeric hub could alter transphosphorylation rates within a holoenzyme, thereby affecting the balance of autophosphorylation outcomes. Using a single-molecule assay for visualization of CaMKII phosphorylation on glass, we show that the balance of autophosphorylation is flipped between CaMKII- and CaMKII-{beta}, the two principal isoforms in the brain. CaMKII-, with a [~]30 residue kinase-hub linker, readily acquires activating autophosphorylation, which we show is resistant to removal by phosphatases. CaMKII-{beta}, with a [~]200 residue kinase-hub linker, is biased towards inhibitory autophosphorylation. Thus, the responsiveness of CaMKII to calcium signals can be tuned by varying the relative levels of the and {beta} isoforms.

biophysics↗

bioRxiv2019

Variation in assembly stoichiometry in non-metazoan homologs of the hub domain of Ca2+/Calmodulin-dependent protein kinase II

The multi-subunit Ca2+/calmodulin-dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is connected by a flexible linker to a C-terminal hub domain that assembles into a 12- or 14-subunit scaffold that displays the kinase domains around it. Studies on CaMKII suggest that the stoichiometry and dynamic assembly/disassembly of hub oligomers may be important for CaMKII regulation. Although CaMKII is a metazoan protein, genes encoding predicted CaMKII-like hub domains, without associated kinase domains, are found in the genomes of some green plants and bacteria. We show that the hub domains encoded by three related green algae, Chlamydomonas reinhardtii, Volvox carteri f. nagarensis, and Gonium pectoral, assemble into 16-, 18-, and 20-subunit oligomers, as assayed by native protein mass spectrometry. These are the largest known CaMKII hub domain assemblies. A crystal structure of the hub domain from Chlamydomonas reinhardtii reveals an 18-subunit organization. We identified four intra-subunit hydrogen bonds in the core of the fold that are present in the Chlamydomonas hub domain, but not in metazoan hubs. When six point mutations designed to recapitulate these hydrogen bonds were introduced into the human CaMKII- hub domain, the mutant protein formed assemblies with 14 and 16 subunits, instead of the normal 12- and 14-subunit assemblies. Our results show that the stoichiometric balance of CaMKII hub assemblies can be shifted readily by small changes in sequence.

biochemistry↗

bioRxiv2018

Switch-like activation of Bruton’s tyrosine kinase by membrane-mediated dimerization

The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that protein-ligand chemical affinities alone generally result in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Brutons tyrosine kinase (Btk) Using fluorescence correlation spectroscopy (FCS) and membrane-binding kinetic measurements, we identify a self-contained phosphatidylinositol (3,4,5)-trisphosphate (PIP3) sensing mechanism that achieves switch-like sensitivity to PIP3 levels, surpassing the intrinsic affinity discrimination of PIP3:PH binding. This mechanism employs multiple PIP3 binding as well as dimerization of Btk on the membrane surface. Mutational studies in live cells confirm that this mechanism is critical for activation of Btk in vivo. These results demonstrate how a single protein module can institute a minimalist coincidence detection mechanism to achieve high-precision discrimination of ligand concentration.

biophysics↗

bioRxiv2018

Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src Homology 2 (SH2) and Phosphotyrosine Binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as phosphosites) must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. In order to understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface-display of peptides, coupled with deep sequencing, to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the -1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins, but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation while maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.

biochemistry↗

bioRxiv2018

Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen

To obtain a comprehensive map of the intrinsic specificities of tyrosine kinase domains, we developed a high-throughput method that uses bacterial surface-display and next-generation sequencing to analyze the specificity of any tyrosine kinase against a library of thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we identified a difference in the electrostatic recognition of substrates between the cytoplasmic Src-family tyrosine kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with the tyrosine kinase ZAP-70 in T cell receptor signaling. The current understanding of substrate recognition by tyrosine kinases emphasizes the role of localization by non-catalytic domains, but our results point to the importance of direct recognition at the kinase active site in fine-tuning specificity. Our method provides a simple approach that leverages next-generation sequencing to readily map the specificity of any tyrosine kinase at the proteome level.

biochemistry↗