Explore the vast range of titles available.

Mitochondrial-derived peptides are encoded by mitochondrial DNA but have biological activity outside mitochondria. Eight of these are encoded by sequences within the mitochondrial 12S and 16S ribosomal genes: humanin, MOTS-c, and the six SHLP peptides, SHLP1-SHLP6. These peptides have various effects in cell culture and animal models, affecting neuroprotection, insulin sensitivity, and apoptosis, and some are secreted, potentially having extracellular signaling roles. However, except for humanin, their importance in normal cell function is unknown. To gauge their importance, their coding sequences in vertebrates have been analyzed for synonymous codon bias. Because they lie in RNA genes, such bias should only occur if their amino acids have been conserved to maintain biological function. Humanin and SHLP6 show strong synonymous codon bias and sequence conservation. In contrast, SHLP1, SHLP2, SHLP3, and SHLP5 show no significant bias and are poorly conserved. MOTS-c and SHLP4 also lack significant bias, but contain highly conserved N-terminal regions, and their biological importance cannot be ruled out. An additional potential mitochondrial-derived peptide sequence was discovered preceding SHLP2, named SHLP2b, which also contains a highly conserved N-terminal region with synonymous codon bias.

Squash is an RNA aptamer that strongly activates the fluorescence of small-molecule analogs of the fluorophore of green fluorescent protein (GFP). Unlike other fluorogenic aptamers, isolated de novo from random-sequence RNA, Squash was evolved from the bacterial adenine riboswitch to leverage its optimized in vivo folding and stability. We now report the 2.7-Å resolution cocrystal structure of fluorophore-bound Squash, revealing that while the overall fold of the riboswitch is preserved, the architecture of the ligand-binding core is dramatically transformed. Unlike previously characterized aptamers that activate GFP-derived fluorophores, Squash does not harbor a G-quadruplex, sandwiching its fluorophore between a base triple and a noncanonical base quadruple in a largely apolar pocket. The expanded structural core of Squash allows it to recognize unnatural fluorophores that are larger than the simple purine ligand of the parental adenine riboswitch, and suggests that stable RNA scaffolds can tolerate larger variation than has hitherto been appreciated.Truong et al. report crystal structures of Squash, a fluorophore-activating aptamer RNA evolved from the adenine riboswitch. Squash preserves the overall scaffold of the adenine riboswitch, yet has a highly divergent ligand-binding site.

Two proton pump inhibitors, tenatoprazole and esomeprazole, were previously shown to inhibit HIV-1 egress by blocking the interaction between Tsg101, a member of the ESCRT-I complex, and ubiquitin. Here, we deepen our understanding of prazole budding inhibition by studying a range of viruses in the presence of tenatoprazole. Furthermore, we investigate the relationship between the chemistry of prodrug activation and HIV-1 inhibition for diverse prazoles currently on the market. We report that tenatoprazole is capable of inhibiting the replication of members of the enveloped filo, alpha, and herpes virus families but not the flavivirus group and not the non-enveloped poliovirus. Another key finding is that prazole prodrugs must be activated inside the cell, while their rate of activation in vitro correlated to their efficacy in cells. Our study lays the groundwork for future efforts to repurpose prazole-based compounds as antivirals that are both broad-spectrum and selective in nature.

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.A combination of structural and protein-chemistry approaches along with phylogenetic analyses provide insights into the specific activities of mouse tubulin tyrosine ligase-like enzymes as initiases or elongases of glutamylation.

BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized α-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB–BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. Apoptosis inducers: BAX activation site identified The trigger mechanism by which apoptosis-inducing proteins such as BAX become activated remains a matter of vigorous debate. Here, a structural analysis of full length BAX in complex with a peptide derived from its activator BIM reveals a novel and unforeseen interaction site. This does not involve the classic hydrophobic groove reported for inhibitors of apoptosis. The identification of BAX's activation site not only provides mechanistic insights into a cell's demise, but also adds a potential new therapeutic target to the list of apoptosis modulators. A structural analysis of the apoptosis-inducing protein BAX in complex with a peptide derived from its activator BIM reveals an unforeseen interaction site that does not involve the classic hydrophobic groove reported for inhibitors of apoptosis. This identification of BAX's activation site provides mechanistic insights into a cell's demise.

Beetroot is a homodimeric in vitro selected RNA that binds and activates DFAME, a conditional fluorophore derived from GFP. It is 70% sequence-identical to the previously characterized homodimeric aptamer Corn, which binds one molecule of its cognate fluorophore DFHO at its interprotomer interface. We have now determined the Beetroot-DFAME co-crystal structure at 1.95 Å resolution, discovering that this RNA homodimer binds two molecules of the fluorophore, at sites separated by ~30 Å. In addition to this overall architectural difference, the local structures of the non-canonical, complex quadruplex cores of Beetroot and Corn are distinctly different, underscoring how subtle RNA sequence differences can give rise to unexpected structural divergence. Through structure-guided engineering, we generated a variant that has a 12-fold fluorescence activation selectivity switch toward DFHO. Beetroot and this variant form heterodimers and constitute the starting point for engineered tags whose through-space inter-fluorophore interaction could be used to monitor RNA dimerization. The recently discovered aptamer Beetroot is a homodimeric RNA that binds and activates DFAME, a conditional, red-shifted fluorophore derived from GFP. Here the authors determine the Beetroot-DFAME co-crystal structure, which is distinctively different from that of similar RNA aptamer Corn.

In isotropic solution, internuclear dipolar couplings average to zero as a result of rotational diffusion. By dissolving macromolecules in a dilute aqueous nematic discotic liquid-crystalline medium containing widely spaced magnetically oriented particles, a tunable degree of solute alignment with the magnetic field can be created while retaining the high resolution and sensitivity of the regular isotropic nuclear magnetic resonance (NMR) spectrum. Dipolar couplings between $^1$H-$^1$H, $^1$H-$^{13}$C, $^1$H-$^{15}$N, and $^{13}$C-$^{13}$C pairs in such an oriented macromolecule no longer average to zero, and are readily measured. Distances and angles derived from dipolar couplings in human ubiquitin are in excellent agreement with its crystal structure. The approach promises to improve the accuracy of structures determined by NMR, and extend the size limit.

HIV-1 replication requires Tsg101, a component of cellular endosomal sorting complex required for transport (ESCRT) machinery. Tsg101 possesses an ubiquitin (Ub) E2 variant (UEV) domain with a pocket that can bind PT/SAP motifs and another pocket that can bind Ub. The PTAP motif in the viral structural precursor polyprotein, Gag, allows the recruitment of Tsg101 and other ESCRTs to virus assembly sites where they mediate budding. It is not known how or even whether the UEV Ub binding function contributes to virus production. Here, we report that disruption of UEV Ub binding by commonly used drugs arrests assembly at an early step distinct from the late stage involving PTAP binding disruption. NMR reveals that the drugs form a covalent adduct near the Ub-binding pocket leading to the disruption of Ub, but not PTAP binding. We conclude that the Ub-binding pocket has a chaperone function involved in bud initiation. Tsg101 is a component of the host cellular ESCRT machinery and is required for HIV-1 replication. Here, the authors show that disruption of ubiquitin binding of the Tsg101 UEV domain through commonly used drugs arrests virus assembly, which might facilitate the development of potent HIV inhibitors.

Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand–protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous 1H-15 N, 1H-13C SESAME based pulse scheme for the rapid acquisition of 1HC/N-R2 relaxation rates for the determination of backbone and sidechain PREs of proteins. The 1HN-R2 rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R2 = 0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.

The Toll/IL-1 receptor (TIR) domains are crucial signaling modules during innate immune responses involving the Toll-like receptors (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 (MyD88) is a central TIR domain-containing adapter molecule responsible for nearly all TLR-mediated signaling and is targeted by a TIR domain-containing protein C (TcpC) from virulent uropathogenic Escherichia coli , a common human pathogen. The mechanism of such molecular antagonism has remained elusive. We present the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops as well as the Poc site. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnish a framework for the design of novel therapeutic agents that modulate immune activation.