Stable m7G Cap-Distal 5'UTR Hairpin Structure Mediates Distinct 40S and 60S Binding Dynamics

The 5' untranslated region (UTR) of diverse mRNAs contains secondary structures that can influence protein synthesis by modulating the initiation step of translation. Studies support the ability of these structures to inhibit 40S subunit recruitment and scanning, but the dynamics of ribosomal subunit interactions with mRNA remain poorly understood. Here, we developed a reconstituted Saccharomyces cerevisiae cell-free translation system with fluorescently labeled ribosomal subunits. We applied this extract and single-molecule fluorescence microscopy to monitor, in real time, individual 40S and 60S interactions with mRNAs containing 5' UTR hairpin structures with varying thermostability. In comparison to mRNAs containing no or weak 5' UTR hairpins (ΔG >= -5.4 kcal/mol), mRNAs with stable hairpins (ΔG <= -16.5 kcal/mol) showed reduced numbers of 60S recruitment to mRNA, consistent with the expectation of reduced translation efficiency for such mRNAs. Interestingly, such mRNAs showed increased numbers of 40S recruitment events to individual mRNAs but with shortened duration on mRNA. Correlation analysis showed that these unstable 40S binding events were nonproductive for 60S recruitment. Furthermore, although the mRNA sequence is long enough to accommodate multiple 40S, individual mRNAs are predominantly observed to engage with a single 40S at a time, indicating the sequestering of mRNA 5' end by initiating 40S. Altogether, these observations suggest that stable cap-distal hairpins in 5' UTR reduce initiation and translation efficiency by destabilizing 40S-mRNA interactions and promoting 40S dissociation from mRNA. The premature 40S dissociation frees mRNA 5'-end accessibility for new initiation events, but the increased rate of 40S recruitment is insufficient to compensate for the reduction of initiation efficiency due to premature 40S dissociation. This study provides the first single-molecule kinetic characterization of 40S/60S interactions with mRNA during cap-dependent initiation and the modulation of such interactions by cap-distal 5' UTR hairpin structures.

Characterization of subunit interactions in the hetero-oligomeric retinoid oxidoreductase complex

The hetero-oligomeric retinoid oxidoreductase complex (ROC) catalyzes the interconversion of all-trans-retinol and all-trans-retinaldehyde to maintain the steady-state output of retinaldehyde, the precursor of all-trans-retinoic acid that regulates the transcription of numerous genes. The interconversion is catalyzed by two distinct components of the ROC: the NAD(H)-dependent retinol dehydrogenase 10 (RDH10) and the NADP(H)-dependent dehydrogenase reductase 3 (DHRS3). The binding between RDH10 and DHRS3 subunits in the ROC results in mutual activation of the subunits. The molecular basis for their activation is currently unknown. Here, we applied site-directed mutagenesis to investigate the roles of amino acid residues previously implied in subunit interactions in other SDRs to obtain the first insight into the subunit interactions in the ROC. The results of these studies suggest that the cofactor binding to RDH10 subunit is critical for the activation of DHRS3 subunit and vice versa. The C-terminal residues 317–331 of RDH10 are critical for the activity of RDH10 homo-oligomers but not for the binding to DHRS3. The C-terminal residues 291–295 are required for DHRS3 subunit activity of the ROC. The highly conserved C-terminal cysteines appear to be involved in inter-subunit communications, affecting the affinity of the cofactor binding site in RDH10 homo-oligomers as well as in the ROC. Modeling of the ROC quaternary structure based on other known structures of SDRs suggests that its integral membrane-associated subunits may be inserted in adjacent membranes of the endoplasmic reticulum (ER), making the formation and function of the ROC dependent on the dynamic nature of the tubular ER network.

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that present a target for insecticides. However, a better understanding of receptor subunit composition is needed for effective design of insecticides. Peptide neurotoxins are known to block nAChRs by binding to its target subunits. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Dα5, Dα6, Dα7 subunits. Finally, we report the localization of fluorescent tagged endogenous Dα6 during nervous system development. Taken together this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins.

Structural basis for the structural dynamics of human mitochondrial chaperonin mHsp60

Human mitochondrial chaperonin mHsp60 is essential for mitochondrial function by assisting folding of mitochondrial proteins. Unlike the double-ring bacterial GroEL, mHsp60 exists as a heptameric ring that is unstable and dissociates to subunits. The structural dynamics has been implicated for a unique mechanism of mHsp60. We purified active heptameric mHsp60, and determined a cryo-EM structure of mHsp60 heptamer at 3.4 Å. Of the three domains, the equatorial domains contribute most to the inter-subunit interactions, which include a four-stranded β sheet. Our structural comparison with GroEL shows that mHsp60 contains several unique sequences that directly decrease the sidechain interactions around the β sheet and indirectly shorten β strands by disengaging the backbones of the flanking residues from hydrogen bonding in the β strand conformation. The decreased inter-subunit interactions result in a small inter-subunit interface in mHsp60 compared to GroEL, providing a structural basis for the dynamics of mHsp60 subunit association. Importantly, the unique sequences are conserved among higher eukaryotic mitochondrial chaperonins, suggesting the importance of structural dynamics for eukaryotic chaperonins. Our structural comparison with the single-ring mHsp60-mHsp10 shows that upon mHsp10 binding the shortened inter-subunit β sheet is restored and the overall inter-subunit interface of mHsp60 increases drastically. Our structural basis for the mHsp10 induced stabilization of mHsp60 subunit interaction is consistent with the literature that mHsp10 stabilizes mHsp60 quaternary structure. Together, our studies provide structural bases for structural dynamics of the mHsp60 heptamer and for the stabilizing effect of mHsp10 on mHsp60 subunit association.

Potent macrocycle inhibitors of the human SAGA deubiquitinating module

The SAGA complex is a transcriptional coactivator that plays multiple roles in activating transcription and is conserved from yeast to humans. One of SAGAs activities is the removal of ubiquitin from histone H2B-K120 by the deubiquitinating module (DUBm), a four-protein subcomplex containing the catalytic subunit, USP22, bound to three proteins that are required for catalytic activity and targeting to nucleosomes. Overexpression of USP22 is correlated with cancers with a poor prognosis that are resistant to available therapies. We used the RaPID (Random non-standard Peptides Integrated Discovery) system to identify cyclic peptides that are potent and highly specific inhibitors of USP22. Peptide binding did not impact the overall integrity of the DUBm complex as judged by small-angle x-ray scattering, indicating that the inhibitors do not disrupt subunit interactions required for USP22 activity. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. The macrocycle inhibitors we have identified in this work thus exhibit favorable drug-like properties and constitute, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module.

Disrupting inter-subunit interactions favors channel opening in P2X2 receptors

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes typically assemble as homo- or heterotrimeric complexes and they contribute to numerous physiological functions, including nociception, inflammation and hearing. Both the overall structure of P2XRs and the details of how ATP is coordinated at the subunit interface are well established. By contrast, little is known about how inter-subunit interactions in the ECD contribute to channel function. Here we investigate both single and double mutants at the subunit interface of rP2X2Rs using electrophysiological and biochemical approaches. Our data demonstrate that the vast majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity and that double mutants at the subunit interface show significant energetic coupling, especially if the mutations are located in close proximity. Overall, we show that inter-subunit interactions, as well as possibly interactions in other parts of the receptor, stabilize WT rP2X2Rs in the closed state. This suggests that, unlike other ligand-gated ion channels, P2X2 receptors have not evolved for an intrinsically low threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

N-terminal extensions strengthen hydrophobic inter-subunit interactions between HU’s C-terminal domains to frustrate heterodimer formation

HU is a nucleoid-associated protein (NAP) that helps bacterial chromosomal DNA to remain compact. Escherichia coli contains two homologs of HU that are ~ 70 % identical: HU-A and HU-B. The early log phase, late log phase, and stationary phase of E. coli growth are reported to be dominated, respectively, by HU-AA homodimers, HU-BB homo-dimers, and HU-AB heterodimers. Here, we show that the formation of HU-AB heterodimers occurs to a much lower degree in HU chains that have a displaced N-terminus, whether through addition of an N-terminal affinity (polyhistidine) tag, or fusion of a fluorescent protein. A combination of mass spectrometry, spectroscopy, chromatography, and electrophoresis (exploring glutaraldehyde crosslinking of subunits) was used to study the mixing, co-expression, unfolding and refolding of HU-AA and HU-BB homodimers. The data suggests that, in HU polypeptides with N-terminal extension, whereas inter-subunit contacts between the alpha helical N-terminal domains (NTDs) undergo facile unfolding and dissociation, inter-subunit contacts between the beta sheet- and IDR-dominated C-terminal domains (CTDs) fail to do so, due to persistence of hydrophobic inter-subunit interactions between two beta sheets. This persistence causes HU to remain nominally dimeric even after substantive unfolding, and frustrates subunit exchange and heterodimer formation.

Structures of human dual oxidase 1 complex in low-calcium and high-calcium states

Dual oxidases (DUOXs) produce hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen. They are involved in many crucial biological processes and human diseases, especially in thyroid diseases. DUOXs are protein complexes co-assembled from the catalytic DUOX subunits and the auxiliary DUOXA subunits and their activities are regulated by intracellular calcium concentrations. Here, we report the cryo-EM structures of human DUOX1-DUOXA1 complex in both high-calcium and low-calcium states. These structures reveal the DUOX1 complex is a symmetric 2:2 hetero-tetramer stabilized by extensive inter-subunit interactions. Substrate NADPH and cofactor FAD are sandwiched between transmembrane domain and the cytosolic dehydrogenase domain of DUOX. In the presence of calcium ions, intracellular EF-hand modules might enhance the catalytic activity of DUOX by stabilizing the dehydrogenase domain in a conformation that allows electron transfer.