SUMMARYAmino-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this co-translational modification, the heterodimeric NatA complex harbors the most diversity for substrate selection and modifies the majority of all amino-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100 kDa holo-NatA complex from Schizosaccharomyces pombe in the absence and presence of a bisubstrate peptide-CoA conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit. The NatA-Naa15p auxiliary subunit contains 13 TPR motifs and adopts a ring-like topology that wraps around the NatA-Naa10p subunit, an interaction that alters the Naa10p active site for substrate-specific acetylation. These studies have implications for understanding the mechanistic details of other NAT complexes and how regulatory subunits modulate the activity of the broader family of protein acetyltransferases.

The CFH group, a potential surrogate for the OH group, can act as an unusual hydrogen bond donor, as confirmed by crystallographic, spectroscopic, and computational methods. Here, we demonstrate the bioisosterism of the OH and CFH groups and the important roles of CF-H···O hydrogen bonds in influencing intermolecular interactions and conformational preferences. Experimental evidence, corroborated by theory, reveals the distinctive nature of CFH hydrogen bonding interactions relative to their normal OH hydrogen bonding counterparts.

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.AMPA receptors | zinc | ZnT3 | synaptic plasticity | auditory T he development, function, and experience-dependent plasticity of the mammalian brain depend on the refined neuronal interactions that occur in synapses. In the majority of excitatory synapses, the release of the neurotransmitter glutamate from presynaptic neurons opens transmembrane ion channels in postsynaptic neurons, the ionotropic glutamate receptors, thereby generating the flow of excitatory signaling in the brain. As a result, these receptors play a fundamental role in normal function and development of the brain, and they are also involved in many brain disorders (1).The ionotropic glutamate receptor family consists of AMPA, kainate, and NMDA receptors (NMDARs). Although kainate receptor-mediated excitatory postsynaptic responses occur in a few central synapses (2), AMPA receptors (AMPARs) and NMDARs are localized in the postsynaptic density of the vast majority of glutamatergic synapses in the brain, mediating most of excitatory neurotransmission (1). NMDAR function is regulated by a wide spectrum of endogenous allosteric neuromodulators that fine-tune synaptic responses (3-5); however, much less is known about endogenous AMPAR neuromodulators [(1, 5), but see refs. 6 and 7]. Recent structural studies revealed that the amino terminal domain (ATD) and ligand-binding domain (LBD) are tightly packed in NMDARs but not AMPARs (8-10). These structural differences explain some of the functional differences in allosteric modulation between AMPARs and NMDARs, such as why the ATD of NMDARs, unlike that of AMPARs, modulates function and contains numerous binding sites for allosteric regulators. Nonetheless, given the importance of fine-tuning both synaptic AMPAR and NMDAR responses for brain function, it is puzzling that there is not much evidence for endogenous, extracellular AMPAR modulation. The discovery and establishment of endogenous AMPAR modulators is crucial both for understanding ionotropic glutamate receptor signaling and for developing therapeutic agents for the treatment of AMPAR-related disorders, such as d...

Decreasing the size of spectroscopic probes can afford higher-resolution structural information from fluorescence experiments. Therefore, we have developed p-cyanophenylalanine (Cnf) and backbone thioamides as a fluorophore/quencher pair. Through the examination of a series of thiopeptides, we have determined the working distance for this pair to be 8-30 Å. We have also carried out a proof-of-principle protein-folding experiment in which a Cnf/thioamide-labeled version of villin headpiece HP35 was thermally unfolded while the Cnf/thioamide distance was monitored by fluorescence. For a given protein, thioamide substitutions could be used to track motions with a much greater number of measurements than for current fluorescence probes, providing a dense array of data with which to model conformational changes.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in E. coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here, we report the synthesis of Acd in 87 % yield over five steps from Tyr, and the identification of an Acd synthetase by screening candidate enzymes previously evolved from M. janaschii Tyr synthetase for unnatural amino acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with select natural amino acids and Förster resonance energy transfer (FRET) interactions with common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu3+ to monitor protein folding and binding interactions.