Architecture and assembly of scaffolded signaling complexes
Dynamic, scaffolded, multi-protein complexes are hallmarks of sophisticated cellular signaling systems. The higher-order architecture of these signaling complexes confers sensitivity, adaptability, control, and crosstalk. Signaling complex architecture is especially notable in the nervous system, where dynamic post-synaptic protein assemblies underlie the molecular origins of learning and memory. Moreover, post-translational modifications within the signaling complexes induce conformational changes to modulate reorganization and retargeting. Understanding the dynamic architecture of these protein assemblies is crucial because disruptions in this system are implicated in neurological disorders such as Parkinson’s disease, autism spectrum disorders, depression, and schizophrenia.
Our lab employs mass spectrometry and chemical tools to probe the interactions and post-translational modifications that contribute to synaptic signaling strength and plasticity. Among these techniques, we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map protein—protein interactions and conformational changes in dynamic signaling complexes. In addition, we employ diverse chemical tools for probing protein—protein interactions, such as crosslinkers, artificial amino acids, and spectroscopic tags. By integrating maps of protein interaction surfaces with the structures of the protein components, we can devise models of the higher-order architecture of the signaling assemblies. Furthermore, we can test how post-translational modifications and conformational changes remodel the architecture. Our targets of inquiry include kinases, trafficking chaperones, nitric oxide signaling components, and the scaffolding proteins that assemble these diverse signaling effectors together at the synaptic membrane.
Neuronal NO Signaling
The diatomic gas nitric oxide (NO) serves dual physiological roles as both a signaling molecule and an inflammation agent. Nitric oxide synthases (NOS) are large, multi-domain hemoproteins responsible for production of NO. Emitted at minute concentrations, NO is a membrane-permeable paracrine signaling agent. Generated at high levels, NO acts as a potent cytotoxic weapon of the immune system. As a fairly reactive free radical, NO production is tightly regulated. NOS regulation is especially important for synaptic NO signaling. NO is uniquely well-suited for local, omnidirectional neurotransmission because it is a short-lived, freely diffusible gas. Unlike classical neurotransmitters, NO cannot be stored in vesicles. Control over NO signaling is exerted by regulating localization and activation. Strict regulation of NO production is vital, as NO surges are a prominent mediator of excitotoxicity.
Excitatory synapses exhibit characteristic proteinaceous microcompartments at the post-synaptic membrane known as the post-synaptic density (PSD). The PSD is richly organized into multi-protein complexes composed of glutamate receptors, scaffolding proteins, kinases, cytoskeleton, and G-protein signaling effectors. The dynamic reorganizations of the higher-order architecture and composition of PSD signaling complexes underlies synaptic plasticity, learning, and memory. Dysfunction or disruption of PSD complexes is implicated in neurological disorders such as Alzheimer’s disease, Parkinson’s disease, autism spectrum disorders, depression, and schizophrenia. Transmembrane glutamate receptor/ion channels (NMDAR and AMPAR) form the functional core of the PSD. The dynamic trafficking of receptors into and out of the PSD ultimately controls the strength of synaptic connections. Scaffolding proteins (e.g., PSD-95, GRIP1, Shank) corral these receptors along with signaling effectors (e.g., CaMKII, Ras, SynGAP, nNOS) and cytoskeleton to actively remodel the PSD and tune neuronal activity.
Illuminating the architecture of these scaffolded PSD signaling complexes remains a frontier goal in molecular neurobiology. Nevertheless, synaptic signaling complexes are large and highly dynamic, problematic targets for structural biology. To address the challenges of exploring the architecture of PSD scaffolded signaling complexes, we apply a battery of protein footprinting, chemical biology, and proteomics approaches.