Lynx1 and the β2V287L mutation affect the stoichiometry of the α4β2 nicotinic acetylcholine receptor

GPI-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx affects assembly of nascent α4 and β2 subunits, and alters the stoichiometry of the population that reaches the plasma membrane. Additionally, these data suggest that lynx1 alters nAChR stoichiometry primarily through this intracellular interaction, rather than via effects on plasma membrane nAChRs. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any GPI-anchored protein, could act within the cell to alter assembly of multi-subunit protein.

Investigating the Role of the GET3-GET4/GET5 Interaction During Tail-Anchor Protein Targeting

The proper targeting of membrane proteins is essential to the viability of all cells. Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET pathway (Guided Entry of TA proteins). In the yeast pathway, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the central targeting factor, the Get3 ATPase. Although binding of Get4/5 to Get3 is critical for efficient TA targeting, the mechanisms by which Get4 regulates Get3 are unknown. To understand the molecular basis of Get4 function, we used a combination of structural biology, biochemistry, and cell biology. Get4/5 binds across the Get3 dimer interface, in an orientation only compatible with a closed Get3, providing insight into the role of nucleotide in complex formation. Additionally, this structure reveals two functionally distinct binding interfaces for anchoring and ATPase regulation, and loss of the regulatory interface leads to strong defects in vitro and in vivo. Additional crystal structures of the Get3-Get4/5 complex give rise to an alternate conformation, which represents an initial binding interaction mediated by electrostatics that facilitates the rate of subsequent inhibited complex formation. This interface is supported by an in-depth kinetic analysis of the Get3-Get4/5 interaction confirming the two-step complex formation. These results allow us to generate a refined model for Get4/5 function in TA targeting.

Design, Synthesis, and Study of Novel Platforms for Iron-N2 Chemistry and Photoinduced, Copper-mediated C-N Bond Formation

Several new ligand platforms designed to support iron dinitrogen chemistry have been developed. First, we report Fe complexes of a tris(phosphino)alkyl (CP<sup>iPrsup><sub>3sub>) ligand featuring an axial carbon donor intended to conceptually model the interstitial carbide atom of the nitrogenase iron-molybdenum cofactor (FeMoco). It is established that in this scaffold, the iron center binds dinitrogen trans to the C<sub>alkylsub> anchor in three structurally characterized oxidation states. Fe-C<sub>alkylsub> lengthening is observed upon reduction, reflective of significant ionic character in the Fe-C<sub>alkylsub> interaction. The anionic (CP<sup>iPrsup><sub>3sub>)FeN<sub>2sub><sup>-sup> species can be functionalized by a silyl electrophile to generate (CP<sup>iPrsup><sub>3sub>)Fe-N<sub>2sub>SiR<sub>3sub>. This species also functions as a modest catalyst for the reduction of N<sub>2sub> to NH<sub>3sub>. Next, we introduce a new binucleating ligand scaffold that supports an Fe(μ-SAr)Fe diiron subunit that coordinates dinitrogen (N<sub>2sub>-Fe(μ-SAr)Fe-N<sub>2sub>) across at least three oxidation states (Fe<sup>IIsup>Fe<sup>IIsup>, Fe<sup>IIsup>Fe<sup>Isup>, and Fe<sup>Isup>Fe<sup>Isup>). Despite the sulfur-rich coordination environment of iron in FeMoco, synthetic examples of transition metal model complexes that bind N<sub>2sub> and also feature sulfur donor ligands remain scarce; these complexes thus represent an unusual series of low-valent diiron complexes featuring thiolate and dinitrogen ligands. The (N<sub>2sub>-Fe(μ-SAr)Fe-N<sub>2sub>) system undergoes reduction of the bound N<sub>2sub> to produce NH<sub>3sub> (~50% yield) and can efficiently catalyze the disproportionation of N<sub>2sub>H<sub>4sub> to NH<sub>3sub> and N<sub>2sub>. The present scaffold also supports dinitrogen binding concomitant with hydride as a co-ligand. Next, inspired by the importance of secondary-sphere interactions in many metalloenzymes, we present complexes of iron in two new ligand scaffolds ([SiP<sup>NMesup><sub>3sub>] and [SiP<sup>iPrsup><sub>2sub>P<sup>NMesup>]) that incorporate hydrogen-bond acceptors (tertiary amines) which engage in interactions with nitrogenous substrates bound to the iron center (NH<sub>3sub> and N<sub>2sub>H<sub>4sub>). Cation binding is also facilitated in anionic Fe(0)-N<sub>2sub> complexes. While Fe-N<sub>2sub> complexes of a related ligand ([SiP<sup>iPrsup><sub>3sub>]) lacking hydrogen-bond acceptors produce a substantial amount of ammonia when treated with acid and reductant, the presence of the pendant amines instead facilitates the formation of metal hydride species.

Additionally, we present the development and mechanistic study of copper-mediated and copper-catalyzed photoinduced C-N bond forming reactions. Irradiation of a copper-amido complex, ((m-tol)<sub>3sub>P)<sub>2sub>Cu(carbazolide), in the presence of aryl halides furnishes N-phenylcarbazole under mild conditions. The mechanism likely proceeds via single-electron transfer from an excited state of the copper complex to the aryl halide, generating an aryl radical. An array of experimental data are consistent with a radical intermediate, including a cyclization/stereochemical investigation and a reactivity study, providing the first substantial experimental support for the viability of a radical pathway for Ullmann C-N bond formation. The copper complex can also be used as a precatalyst for Ullmann C-N couplings. We also disclose further study of catalytic C<sub>alkylsub>-N couplings using a CuI precatalyst, and discuss the likely role of [Cu(carbazolide)<sub>2sub>]<sup>-sup> and [Cu(carbazolide)<sub>3sub>]<sup>-sup> species as intermediates in these reactions.

Finally, we report a series of four-coordinate, pseudotetrahedral P<sub>3sub>Fe<sup>IIsup>-X complexes supported by tris(phosphine)borate ([PhBP<sub>3sub>Fe<sup>Rsup>]<sup>-sup>) and phosphiniminato X-type ligands (-N=PR'<sub>3sub>) that in combination tune the spin-crossover behavior of the system. Low-coordinate transition metal complexes such as these that undergo reversible spin-crossover remain rare, and the spin equilibria of these systems have been studied in detail by a suite of spectroscopic techniques.