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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2021)

Synaptic plasticity underlies all learning and memory and is a cornerstone of neurological function. The adaptive and flexible nature of these processes makes them complex and challenging to define with the precision needed to develop quantitative models or effective therapeutics. The main excitatory neurotransmitter in the mammalian nervous system is glutamate which is also critical in setting synaptic strength and plasticity. Glutamate induces synaptic changes through two classes of receptors, ionotropic glutamate receptors and metabotropic glutamate receptors. Ionotropic glutamate receptors are glutamate gated calcium channels which can alter synaptic strength in the time scale of tens of milliseconds to 1 second. Metabotropic glutamate receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) which induce synaptic changes on the time scale of tens of seconds to minutes, hours and even days. This makes mGluRs critical to understanding the molecular induction of synaptic plasticity and very attractive therapeutic targets. Despite much effort put towards developing therapeutics that target mGluRs none have been approved by the FDA. A deeper understanding of mGluR activation will aid in designing more effective therapeutics as well as provide a more precise role of glutamate on synaptic changes.mGluRs are obligate dimers with large N-terminal domains. Each subunit contains a clamshell like glutamate binding domain (LBD), a rigid cysteine rich domain (CRD) and the canonical 7-pass transmembrane domain that defines all GPCRs. Numerous studies of GPCRs have pointed to the fact that these receptors are highly dynamic and that ligand binding changes the time a receptor spends in several distinct conformational states. The last ten years has seen an explosion in the number of high-resolution GPCRs structures. Unfortunately, these only provide a snapshot of a subset of potential receptor conformations and the amount of knowledge on the conformational dynamics of GPCRs has lagged behind. One very promising technique to address the questions surrounding GPCR conformational dynamics is single molecule fluorescence resonance energy transfer (smFRET). smFRET allows for very precise measurement of receptor motions and compliments the wealth of structural information to create a much more complete understanding of GPCRs. To this end I used smFRET to measure and study the conformational motions associated with activation of mGluR homodimers and heterodimers. First I examined the effects of different regions on mGluR2 homodimer activation and mGluR2/3 heterodimer activation which demonstrated a functional role of a LBD interface and the cysteine loop. Next I examined mGluR7, which has an incredibly low glutamate affinity, and found that while mGluR7 homodimers are virtually insensitive to glutamate, mGluR2/7 heterodimers are present in native tissue and are super sensitive to glutamate due to an apo state rotation demonstrating a potential biologic and modulatory role of heterodimerization. Lastly, I examined regions of mGluR7 and find that the lower dimer interface is responsible for very low glutamate sensitivity and that the cysteine bridge flexibility is responsible for the mGluR2/7 super sensitivity to glutamate.

    Cover page: Mechanisms of activation and modulation of homomeric and heteromeric metabotropic glutamate receptors
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2021)
    N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.
      Cover page: Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2023)
      Metabotropic glutamate receptors (mGluRs) are dimeric class C G-protein-coupled receptors that operate in glia and neurons. Glutamate affinity and efficacy vary greatly between the eight mGluRs. The molecular basis of this diversity is not understood. We used single-molecule fluorescence energy transfer to monitor the structural rearrangements of activation in the mGluR ligand binding domain (LBD). In saturating glutamate, group II homodimers fully occupy the activated LBD conformation (full efficacy) but homodimers of group III mGluRs do not. Strikingly, the reduced efficacy of Group III homodimers does not arise from differences in the glutamate binding pocket but, instead, from interactions within the extracellular dimerization interface that impede active state occupancy. By contrast, the functionally boosted mGluR II/III heterodimers lack these interface brakes to activation and heterodimer asymmetry in the flexibility of a disulfide loop connecting LBDs greatly favors occupancy of the activated conformation. Our results suggest that dimerization interface interactions generate substantial functional diversity by differentially stabilizing the activated conformation. This diversity may optimize mGluR responsiveness for the distinct spatio-temporal profiles of synaptic versus extrasynaptic glutamate.
        Cover page: Homo- and hetero-dimeric subunit interactions set affinity and efficacy in metabotropic glutamate receptors.
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2016)
        G protein-coupled receptors (GPCRs) mediate cellular responses to a wide variety of extracellular stimuli. GPCR dimerization may expand signaling diversity and tune functionality, but little is known about the mechanisms of subunit assembly and interaction or the signaling properties of heteromers. Using single-molecule subunit counting on class C metabotropic glutamate receptors (mGluRs), we map dimerization determinants and define a heterodimerization profile. Intersubunit fluorescence resonance energy transfer measurements reveal that interactions between ligand-binding domains control the conformational rearrangements underlying receptor activation. Selective liganding with photoswitchable tethered agonists conjugated to one or both subunits of covalently linked mGluR2 homodimers reveals that receptor activation is highly cooperative. Strikingly, this cooperativity is asymmetric in mGluR2/mGluR3 heterodimers. Our results lead to a model of cooperative activation of mGluRs that provides a framework for understanding how class C GPCRs couple extracellular binding to dimer reorganization and G protein activation.
          Cover page: Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2024)
          N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties. To understand this diversity, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure the conformations of the ligand binding domain and modulatory amino-terminal domain of the common GluN1 subunit in receptors with different GluN2 subunits. Our results demonstrate a strong influence of the GluN2 subunits on GluN1 rearrangements, both in non-agonized and partially agonized activation intermediates, which have been elusive to structural analysis, and in the fully liganded state. Chimeric analysis reveals structural determinants that contribute to these subtype differences. Our study provides a framework for understanding the conformational landscape that supports highly divergent levels of activity, desensitization, and agonist potency in receptors with different GluN2s and could open avenues for the development of subtype-specific modulators.
            Cover page: Subtype-specific conformational landscape of NMDA receptor gating.
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2019)
            Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors that operate at synapses. Macroscopic and single molecule FRET to monitor structural rearrangements in the ligand binding domain (LBD) of the mGluR7/7 homodimer revealed it to have an apparent affinity ~4000-fold lower than other mGluRs and a maximal activation of only ~10%, seemingly too low for activation at synapses. However, mGluR7 heterodimerizes, and we find it to associate with mGluR2 in the hippocampus. Strikingly, the mGluR2/7 heterodimer has high affinity and efficacy. mGluR2/7 shows cooperativity in which an unliganded subunit greatly enhances activation by agonist bound to its heteromeric partner, and a unique conformational pathway to activation, in which mGluR2/7 partially activates in the Apo state, even when its LBDs are held open by antagonist. High sensitivity and an unusually broad dynamic range should enable mGluR2/7 to respond to both glutamate transients from nearby release and spillover from distant synapses.
              Cover page: Conformational pathway provides unique sensitivity to a synaptic mGluR
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2016)
              CTP synthetases catalyze the last step of pyrimidine biosynthesis and provide the sole de novo source of cytosine-containing nucleotides. As a central regulatory hub, they are regulated by ribonucleotide and enzyme concentration through ATP and UTP substrate availability, CTP product inhibition, GTP allosteric modification, and quaternary structural changes including the formation of CTP-inhibited linear polymers (filaments). Here, we demonstrate that nicotinamide redox cofactors are moderate inhibitors of Escherichia coli CTP synthetase (EcCTPS). NADH and NADPH are the most potent, and the primary inhibitory determinant is the reduced nicotinamide ring. Although nicotinamide inhibition is noncompetitive with substrates, it apparently enhances CTP product feedback inhibition and GTP allosteric regulation. Further, CTP and GTP also enhance each other's effects, consistent with the idea that NADH, CTP, and GTP interact with a common intermediate enzyme state. A filament-blocking mutation that reduces CTP inhibitory effects also reduced inhibition by GTP but not NADH. Protein-concentration effects on GTP inhibition suggest that, like CTP, GTP preferentially binds to the filament. All three compounds display nearly linear dose-dependent inhibition, indicating a complex pattern of cooperative interactions between binding sites. The apparent synergy between inhibitors, in consideration with physiological nucleotide concentrations, points to metabolically relevant inhibition by nicotinamides, and implicates cellular redox state as a regulator of pyrimidine biosynthesis.
                Cover page: Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2023)
                Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.
                  Cover page: Negative allosteric modulation of the glucagon receptor by RAMP2
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2014)
                  CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.
                    Cover page: Large-scale filament formation inhibits the activity of CTP synthetase.
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2013)
                    Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN >> NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.
                      Cover page: Quantitative analysis of TALE–DNA interactions suggests polarity effects
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