Gating mechanisms of voltage-gated proton channels.

Hv1 is a voltage-gated proton-selective channel that plays critical parts in host defense, sperm motility, and cancer progression. Hv1 contains a conserved voltage-sensor domain (VSD) that is shared by a large family of voltage-gated ion channels, but it lacks a pore domain. Voltage sensitivity and proton conductivity are conferred by a unitary VSD that consists of four transmembrane helices. The architecture of Hv1 differs from that of cation channels that form a pore in the center among multiple subunits (as in most cation channels) or homologous repeats (as in voltage-gated sodium and calcium channels). Hv1 forms a dimer in which a cytoplasmic coiled coil underpins the two protomers and forms a single, long helix that is contiguous with S4, the transmembrane voltage-sensing segment. The closed-state structure of Hv1 was recently solved using X-ray crystallography. In this article, we discuss the gating mechanism of Hv1 and focus on cooperativity within dimers and their sensitivity to metal ions.

The Study of Metalloproteome of DNA Viruses: Identification, Functional Annotation, and Diversity Analysis of Viral Metal-Binding Proteins.

Metalloproteins are fundamental to diverse biological processes but still lack extensive investigation in viral contexts. This study reveals the prevalence and functional diversity of metal-binding proteins in DNA viruses. Among a subset of 1432 metalloproteins, zinc and magnesium-binding proteins are notably abundant, indicating their importance in viral biology. Furthermore, significant numbers of proteins binding to iron, manganese, copper, nickel, mercury, and cadmium were also detected. Human-infecting viral proteins displayed a rich landscape of metalloproteins, with MeBiPred (964 proteins) and Pfam (666) yielding the highest numbers. Interestingly, many essential viral proteins exhibited metal-binding capabilities, including polymerases, DNA binding proteins, helicases, dUPTase, thymidine kinase, and various structural and accessory proteins. This study sheds light on the ubiquitous presence of metalloproteins, their functional signatures, subcellular placements, and metal-utilization patterns, providing valuable insights into viral biology. A similar metal utilization pattern was observed in similar functional proteins across the various DNA viruses. Furthermore, these findings provide a foundation for identifying potential drug targets for combating viral infections.

Difluoromethyl-1,3,4-oxadiazoles are slow-binding substrate analog inhibitors of histone deacetylase 6 with unprecedented isotype selectivity.

Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.

Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment.

BACKGROUND: Alpha-2-glycoprotein 1, zinc-binding (ZAG), a secreted protein encoded by the AZGP1 gene, is structurally similar to HLA class I. Despite its presumed immunological function, little is known about its role in tumor immunity. In this study, we thus aimed to determine the relationship between the expression of AZGP1/ZAG and the immunological profiles of breast cancer tissues at both the gene and protein level. METHODS: Using a publicly available gene expression dataset from a large-scale breast cancer cohort, we conducted gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1. We analyzed the correlation between AZGP1 expression and immune cell composition in breast cancer tissues, estimated using CIBERSORTx. Previously, we evaluated the infiltration of 11 types of immune cells for 45 breast cancer tissues using flow cytometry (FCM). ZAG expression was evaluated by immunohistochemistry on these specimens and analyzed for its relationship with immune cell infiltration. The action of ZAG in M1/M2 polarization models using primary cultures of human peripheral blood mononuclear cells (PBMC)-derived macrophage (Mφ) was analyzed based on the expression of M1/M2 markers (CD86, CD80/CD163, MRC1) and HLA class I/II by FCM. RESULTS: AZGP1 expression was negatively correlated with multiple immunological processes and specific immune cell infiltration including Mφ M1 using GSEA and CIBERSORTx. ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in tumor tissues assessed using FCM. In in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I/II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. CONCLUSION: ZAG is suggested to be a novel immunoregulatory factor affecting the Mφ phenotype in breast cancer tissues.

Role of the interaction between troponin T and AMP deaminase by zinc bridge in modulating muscle contraction and ammonia production.

The N-terminal region of troponin T (TnT) does not bind any protein of the contractile machinery and the role of its hypervariability remains uncertain. In this review we report the evidence of the interaction between TnT and AMP deaminase (AMPD), a regulated zinc enzyme localized on the myofibril. In periods of intense muscular activity, a decrease in the ATP/ADP ratio, together with a decrease in the tissue pH, is the stimulus for the activation of the enzyme that deaminating AMP to IMP and NH3 displaces the myokinase reaction towards the formation of ATP. In skeletal muscle subjected to strong tetanic contractions, a calpain-like proteolytic activity produces the removal in vivo of a 97-residue N-terminal fragment from the enzyme that becomes desensitized towards the inhibition by ATP, leading to an unrestrained production of NH3. When a 95-residue N-terminal fragment is removed from AMPD by trypsin, simulating in vitro the calpain action, rabbit fast TnT or its phosphorylated 50-residue N-terminal peptide binds AMPD restoring the inhibition by ATP. Taking in consideration that the N-terminus of TnT expressed in human as well as rabbit white muscle contains a zinc-binding motif, we suggest that TnT might mimic the regulatory action of the inhibitory N-terminal domain of AMPD due to the presence of a zinc ion connecting the N-terminal and C-terminal regions of the enzyme, indicating that the two proteins might physiologically associate to modulate muscle contraction and ammonia production in fast-twitching muscle under strenuous conditions.

Tail-approach based design, synthesis, and molecular modeling of benzenesulfonamides carrying thiadiazole and urea moieties as novel carbonic anhydrase inhibitors.

We synthesized herein 16 compounds (SUT1-SUT16) as potential carbonic anhydrase (CA) inhibitors utilizing the tail-approach design. Based on this strategy, we connected benzenesulfonamide, the zinc-binding scaffold, to different urea moieties with the 1,3,4-thiadiazole ring as a linker. We obtained the target compounds by the reaction of 4-(5-amino-1,3,4-thiadiazol-2-yl)benzenesulfonamide with aryl isocyanates. Upon confirmation of their structures, the compounds were screened for their ability to inhibit the tumor-related human (h) isoforms human carbonic anhydrase (hCA) IX and XII, as well as the physiologically dominant hCA I and II. Most of the molecules demonstrated Ki values ≤ 10 nM with different selectivity profiles. The binding modes of SUT9, SUT10, and SUT5, the most effective inhibitors of hCA II, IX, and XII, respectively, were predicted by molecular docking. SUT16 (4-{5-[3-(naphthalen-1-yl)ureido]-1,3,4-thiadiazol-2-yl}benzenesulfonamide) was found to be the most selective inhibitor of the cancer-associated isoforms hCA IX and XII over the off-target isoforms, hCAI and II. The interaction dynamics and stability of SUT16 within hCA IX and XII were investigated by molecular dynamics simulations as well as dynophore analysis. Based on computational data, increased hydrophobic contacts and hydrogen bonds in the tail part of these molecules within hCA IX and XII were found as favorable interactions leading to effective inhibitors of cancer-related isoforms.

Exploring the Influence of Zinc Ions on the Conformational Stability and Activity of Protein Disulfide Isomerase.

The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

Purification, Characterization, cDNA Cloning, and Bioinformatic Analysis of Zinc-Binding Protein from Magallana hongkongensis.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key ß-barrel, ß-sandwich, ß-α-ß, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Circular permutation at azurin's active site slows down its folding.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn2+-bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn2+-CP protein was much slower than that of the Zn2+-WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn2+-azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn2+-cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn2+- WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes.

A novel loss of function mutation in the PHD domain of the RAG2 gene, affecting zinc-binding affinity.

BACKGROUND: Severe combined immune deficiencies (SCIDs) are genetically heterogeneous disorders that lead to the absence or malfunction of adaptive immune cells, including T- and B-cells. Pathogenic variants in the RAG2 gene are associated with this disease. METHODS: A couple with consanguineous marriage from the Iranian-Azeri-Turkish ethnic group was referred to the genetic lab. Two children of this family died due to SCID disease with symptoms of skin granulomas, lack of developed T- and B-cells, and intact NK cells. To infer their genotypes, DNA samples obtained from the parents were subjected to whole-exome sequencing (WES). RESULTS: WES data analysis revealed that both parents were carriers of a pathogenic variant, NC_000011.10 (NM_000536.4):c.1268G > C, in the RAG2 gene. This variant was absent in our cohort of 400 healthy individuals from the same ethnic group. To gain insight into the consequence of the variant on the protein function, further analysis was performed by applying bioinformatics tools. This study revealed that the replacement of cysteine with serine at the zinc-binding domain diminished the domain's affinity to zinc ion, resulting in the loss of the mutant protein's ability to bind to the recombination signal sequence (RSS). The formation of the RAG2-RSS complex is vital for T- and B-cell development. CONCLUSION: The identification of a novel pathogenic variant, c.1268G > C, revealed that this variant in the zinc-binding domain diminished the affinity of the zinc ion to the mutant protein and consequently led to the loss of its ability to bind to the RSS.

Design and synthesis of some new benzoylthioureido benzenesulfonamide derivatives and their analogues as carbonic anhydrase inhibitors.

The present investigation reports the design and synthesis of three series of benzoylthioureido derivatives bearing either benzenesulfonamide 7a-f, benzoic acid 8a-f or ethylbenzoate 9a-f moieties. The synthesised compounds were screened for their carbonic anhydrase inhibitory activity (CAI) against four isoforms hCA I, II, IX, and XII. Compounds 7a, 7b, 7c, and 7f exhibited a potent inhibitory activity towards hCAI (Kis = 58.20, 56.30, 33.00, and 43.00 nM), respectively compared to acetazolamide (AAZ) and SLC-0111 (Kis = 250.00 and 5080.00 nM). Compounds 7a, 7b, 7c, 7e, and 7f elicited selectivity over h CA II (Kis = 2.50, 2.10, 56.60,39.60 and 39.00 nM) respectively, relative to AAZ and SLC-0111(Kis = 12.10 and 960.00 nM). Also, compounds 7c, 7f, and 9e displayed selectivity against the tumour-associated isoform hCA IX (Kis = 31.20, 30.00 and 29.00 nM) respectively, compared to AAZ and SLC-0111 (Kis = 25.70 and 45.00 nM). Additionally, compounds 8a and 8f revealed a moderate to superior selectivity towards hCAXII (Kis = 17.00 and 11.00 nM) relative to AAZ and SLC-0111(Kis = 5.70 and 45.00 nM). Molecular docking and ADME prediction studies were performed on the most active compounds to shed light on their interaction with the hot spots of the active site of CA isoforms, in addition to prediction of their pharmacokinetic and physicochemical properties.

A Near InfraRed Emissive Chemosensor for Zn2+ and Phosphate Derivatives Based on a Di-(2-picolyl)amine-styrylflavylium Push-Pull Fluorophore.

A new Near InfraRed (NIR) fluorescent chemosensor for metal ions and anions is herein presented. The fluorophore is based on a styrylflavylium dye, a synthetic analogue of the natural anthocyanin family, with a di-(2-picolyl)amine (DPA) moiety as the metal chelating unit. The substitution pattern of the styrylflavylium core (with tertiary amines on positions 7 and 4') shifts the optical properties of the dye towards the NIR region of the electronic spectra, due to a strong push-pull character over the π-conjugated system. The NIR chemosensor is highly sensitive to the presence of Zn2+, which induces a strong CHelation Enhanced Fluorescence (CHEF) effect upon binding to the DPA unit (2.7 fold increase). The strongest competing ion is Cu2+, with a complete fluorescence quenching, while other metals induce lower responses on the optical properties of the chemosensor. Subsequent anion screening of the Zn2+-chemosensor coordination compound has demonstrated a distinct selectivity towards adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP), with high association constants (K ~ 106 M-1) and a strong CHEF effect (2.4 and 2.9 fold fluorescence increase for ATP and ADP, respectively). Intracellular studies with the Zn2+-complexed sensor showed strong luminescence in the cellular membrane of Gram- bacteria (E. coli) and mitochondrial membrane of mammalian cells (A659), which highlights its possible application for intracellular labelling.

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9.

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-ß-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC-tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.

Resolving the zinc binding capacity of honey bee vitellogenin and locating its putative binding sites.

The protein vitellogenin (Vg) plays a central role in lipid transportation in most egg-laying animals. High Vg levels correlate with stress resistance and lifespan potential in honey bees (Apis mellifera). Vg is the primary circulating zinc-carrying protein in honey bees. Zinc is an essential metal ion in numerous biological processes, including the function and structure of many proteins. Measurements of Zn2+ suggest a variable number of ions per Vg molecule in different animal species, but the molecular implications of zinc-binding by this protein are not well-understood. We used inductively coupled plasma mass spectrometry to determine that, on average, each honey bee Vg molecule binds 3 Zn2+ -ions. Our full-length protein structure and sequence analysis revealed seven potential zinc-binding sites. These are located in the ß-barrel and α-helical subdomains of the N-terminal domain, the lipid binding site, and the cysteine-rich C-terminal region of unknown function. Interestingly, two potential zinc-binding sites in the ß-barrel can support a proposed role for this structure in DNA-binding. Overall, our findings suggest that honey bee Vg bind zinc at several functional regions, indicating that Zn2+ -ions are important for many of the activities of this protein. In addition to being potentially relevant for other egg-laying species, these insights provide a platform for studies of metal ions in bee health, which is of global interest due to recent declines in pollinator numbers.

Comparison of three zinc binding groups for HDAC inhibitors - A potency, selectivity and enzymatic kinetics study.

Hydroxamic acid and benzamide are the most commonly used zinc binding group (ZBG) for HDAC inhibitors both in clinic and pre-clinic. Recently, we discovered several analogs of new type HDAC inhibitors with hydrazide as ZBG. Representative compounds displayed high potency, class I HDAC selectivity and excellent pharmacokinetics profile. In this research, we synthesize tool compounds 4 and 6 by modifying the hydroxamic acid of SAHA with benzamide and hydrazide, respectively, and compare the potency, isoform selectivity, binding profile and enzymatic kinetics for the hydroxamate, benzamide and hydrazide-based inhibitors. It is well known that SAHA with hydroxamic acid is a pan-HDAC inhibitor with competitive binding and fast-on/fast-off profile. Compound 6 is a slow-binding class I selective inhibitor with mixed (competitive and non-competitive) binding mode, which is the same as the hydrazide inhibitors in our previous study. Compound 4 is a class I selective, fast-on/fast-off inhibitor with competitive binding mode to HDAC1/2/3, which is different with published benzamide MS275 and 106. Therefore, the kinetics profile of benzamide is not only due to the ZBG, but also rely on the cap and linker groups. To the best of our knowledge, this is the first report to compare the enzymatic profile of three promising ZBGs of HDAC inhibitors.

Investigating hypozincemia and validity of plasma zinc measurements in infected patients.

Hypozincemia is a well-known phenomenon in patients with infection caused by the activation of the acute phase response (APR). Zn status is still based upon plasma Zn levels in venous blood samples. Recent trials have questioned the validity of this measurement in infected patients. The aim of this study was to assess plasma levels of Zn, albumin and Zinc-binding capacity in patients during and following infection. Furthermore, to assess if an assay for albumin-corrected Zn could potentially replace or add knowledge to existing tools for assessment of Zinc-status. A prospective clinical observational trial was conducted. Associations between P-Zn, -Albumin, -Albumin-corrected Zn and Zn binding capacity were analyzed. Analyzes were based upon two venous blood samples drawn during and following infection, respectively. Twenty-three patients admitted to a medical ward showing paraclinical signs of infection were included in the study. Significantly lower levels of Zn and albumin were found during infection compared with the levels post-infection. These findings corresponded to the changes found in Zn binding capacity. About 52% of patients were deemed Zn deficient by plasma Zn levels during infection but after applying the correction for P-Albumin, all patients were found to be within normal ranges of Zn levels. Furthermore, we found no statistically significant difference between albumin-corrected Zn during infection and P-Zn post-infection. The new assay was found to accurately estimate the 'true' Zn levels in infected patients. Based on our findings, we propose albumin-corrected P-Zn as a promising new tool, which may result in more precise diagnostics and treatment.

Diversely substituted sulfamides for fragment-based drug discovery of carbonic anhydrase inhibitors: synthesis and inhibitory profile.

A series of sulfamide fragments has been synthesised and investigated for human carbonic anhydrase inhibition. One of the fragments showing greater selectivity for cancer-related isoforms hCA IX and XII was co-crystalized with hCA II showing significant potential for fragment periphery evolution via fragment growth and linking. These opportunities will be identified in the future via the screening of this fragment structure for co-operative carbonic anhydrase binding with other structurally diverse fragments.[Figure: see text].

Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation.

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR's MCT-1-binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins.

Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3' untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins, which recognizes RNA via the tandem RNA recognition motifs (RRMs). Here we report the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein-protein and protein-RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3' UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.